Targeted modification of rat genome

ABSTRACT

Compositions and methods are provided for modifying a rat genomic locus of interest using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Compositions and methods for generating a genetically modified rat comprising one or more targeted genetic modifications in their germline are also provided. Compositions and methods are provided which comprise a genetically modified rat or rat cell comprising a targeted genetic modification in the rat interleukin-2 receptor gamma locus, the rat ApoE locus, the rat Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. The various methods and compositions provided herein allows for these modified loci to be transmitted through the germline.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/410,252, filed Jan. 19, 2017, which is a divisional applicationof U.S. application Ser. No. 14/254,715, filed Apr. 16, 2014, whichclaims the benefit of U.S. Provisional Application No. 61/812,319, filedApr. 16, 2013, and U.S. Provisional Application No. 61/914,768, filedDec. 11, 2013, all of which are hereby incorporated herein in theirentirety by reference.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named532676SEQLIST.TXT, created on Jun. 25, 2019, and having a size of 15.1kilobytes, and is filed concurrently with the specification. Thesequence listing contained in this ASCII formatted document is part ofthe specification and is herein incorporated by reference in itsentirety.

FIELD OF INVENTION

Isolated non-human totipotent or pluripotent stem cells, in particularrat embryonic stem cells, that are capable of sustaining pluripotencyfollowing one or more serial genetic modifications in vitro, and thatare capable of transmitting the targeted genetic modifications tosubsequent generations through germline. Compositions and methods formodifying a rat genomic locus of interest via bacterial homologousrecombination (BHR) in a prokaryotic cell. Compositions and methods forgenetically modifying a rat genomic locus of interest using a largetargeting vector (LTVEC) in combination with endonucleases. Compositionsand methods for producing a genetically modified rat comprising one ormore targeted genetic modifications.

BACKGROUND OF THE INVENTION

While rats have been regarded as an important animal model system thatcan recapitulate the pathology of various human diseases, including, butnot limited to, cardiovascular (e.g., hypertension), metabolic (e.g.,obesity, diabetes), neurological (e.g., pain pathologies), and a varietyof cancers, the use of rats in modeling human diseases has been limitedas compared to mice, due in part to unavailability ofgermline-transmittable pluripotent rat cells, which can sustain theirpluripotency following a series of genetic modifications in vitro, e.g.,one or more serial electroporations, and due in part to lack ofefficient targeting technologies that allow introduction or deletion oflarge genomic DNA sequences, or replacement of large endogenous genomicDNA sequences with exogenous nucleic acid sequences in pluripotent ratcells.

There is a need in the art for compositions and methods that allowprecise targeted changes in the genome of a rat, which can open orexpand current areas of target discovery and validate therapeutic agentsmore quickly and easily.

SUMMARY

Methods are provided for modifying a genomic locus of interest in apluripotent cell via targeted genetic modification. Such a methodcomprises (a) introducing into the pluripotent cell a large targetingvector (LTVEC) comprising an insert nucleic acid flanked with a 5′homology arm and a 3′ homology arm; and (b) identifying a geneticallymodified pluripotent cell comprising the targeted genetic modificationat the genomic locus of interest, wherein the targeted geneticmodification is capable of being transmitted through the germline.

In one embodiment, the pluripotent cell is derived from a non-humananimal, including, but not limited to, a rodent, a human, a rat, amouse, a hamster, a rabbit, a pig, a bovine, a deer, a sheep, a goat, achicken, a cat, a dog, a ferret, a primate (e.g., marmoset, rhesusmonkey), a domesticated mammal or an agricultural mammal, or any otherorganism of interest.

In one embodiment, the pluripotent cell is a non-human pluripotent cell.In one embodiment, the non-human pluripotent cell is a mammalianpluripotent cell. In one embodiment, the mammalian pluripotent cell is arodent pluripotent cell. In one embodiment, the rodent pluripotent cellis a rat or mouse pluripotent cell. In one embodiment, the pluripotentcell is a human induced pluripotent stem (iPS) cell.

In one embodiment, the pluripotent cell is a non-human fertilized egg atthe single cell stage. In one embodiment, the non-human fertilized eggis a mammalian fertilized egg. In one embodiment, the mammalianfertilized egg is a rodent fertilized egg at the single cell stage. Inone embodiment, the mammalian fertilized egg is a rat or mousefertilized egg at the single cell stage.

In some embodiments, the sum total of the 5′ and the 3′ homology arms ofthe LTVEC is at least 10 kb. In some embodiments, the sum total of the5′ and the 3′ homology arms of the LTVEC is at least 10 kb but less than100 kb or the sum total of the 5′ and the 3′ homology arms of the LTVECis at least 10 kb but less than 150 kb. In other embodiments, the sizeof the sum total of the total of the 5′ and 3′ homology arms of theLTVEC is about 10 kb to about 150 kb, about 10 kb to about 100 kb, about10 kb to about 75 kb, about 20 kb to about 150 kb, about 20 kb to about100 kb, about 20 kb to about 75 kb, about 30 kb to about 150 kb, about30 kb to about 100 kb, about 30 kb to about 75 kb, about 40 kb to about150 kb, about 40 kb to about 100 kb, about 40 kb to about 75 kb, about50 kb to about 150 kb, about 50 kb to about 100 kb, or about 50 kb toabout 75 kb, about 10 kb to about 30 kb, about 20 kb to about 40 kb,about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80 kb toabout 100 kb, about 100 kb to about 120 kb, or from about 120 kb toabout 150 kb. In one embodiment, the size of the deletion is the same orsimilar to the size of the sum total of the 5′ and 3′ homology arms ofthe LTVEC.

In some such embodiments, the targeted genetic modification isbiallelic.

In some embodiments, the pluripotent cell is a pluripotent rat cell. Inone embodiment, the pluripotent rat cell is a rat embryonic stem cell.In one embodiment, the pluripotent rat cell is derived from a DA strainor an ACI strain. In some embodiments, the pluripotent rat cell ischaracterized by expression of at least one pluripotency markercomprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIFreceptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combinationthereof. In some such methods, the pluripotent rat cell is characterizedby one of more of the following characteristics: (a) lack of expressionof one or more pluripotency markers comprising c-Myc, Ecat1, and/orRexo1; (b) lack of expression of mesodermal markers comprising Brachyuryand/or Bmpr2; (c) lack of expression of one or more endodermal markerscomprising Gata6, Sox17 and/or Sox7; or (d) lack of expression of one ormore neural markers comprising Nestin and/or Pax6. Such methods providethat the sum total of the 5′ and the 3′ homology arms of the LTVEC isfrom about 10 kb to about 30 kb, from about 20 kb to about 40 kb, fromabout 40 kb to about 60 kb, from about 60 kb to about 80 kb, or fromabout 80 kb to about 100 kb, from about 100 kb to about 120 kb, fromabout 120 kb to about 150 kb, or from about 10 kb but less than about150 kb. In some embodiments, the sum total of the 5′ and the 3′ homologyarms of the LTVEC is from about 16 Kb to about 100 Kb. In otherembodiments, the size of the sum total of the total of the 5′ and 3′homology arms of the LTVEC is about 10 kb to about 150 kb, about 10 kbto about 100 kb, about 10 kb to about 75 kb, about 20 kb to about 150kb, about 20 kb to about 100 kb, about 20 kb to about 75 kb, about 30 kbto about 150 kb, about 30 kb to about 100 kb, about 30 kb to about 75kb, about 40 kb to about 150 kb, about 40 kb to about 100 kb, about 40kb to about 75 kb, about 50 kb to about 150 kb, about 50 kb to about 100kb, about 50 kb to about 75 kb, about 10 kb to about 30 kb, about 20 kbto about 40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb,about 80 kb to about 100 kb, about 100 kb to about 120 kb, or from about120 kb to about 150 kb. In one embodiment, the size of the deletion isthe same or similar to the size of the sum total of the 5′ and 3′homology arms of the LTVEC.

The methods further provide that targeted genetic modification (a)comprises a replacement of an endogenous rat nucleic acid sequence witha homologous or an orthologous mammalian nucleic acid sequence; (b)comprises a deletion of an endogenous rat nucleic acid sequence; (c)comprises a deletion of an endogenous rat nucleic acid sequence, whereinthe deletion ranges from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200kb, from about 200 kb to about 300 kb, from about 300 kb to about 400kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb,from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, fromabout 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (d)comprises an exogenous nucleic acid sequence ranging from about 5 kb toabout 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,from about 80 kb to about 100 kb, from about 100 kb to about 150 kb,from about 150 kb to about 200 kb, from about 200 kb to about 250 kb,from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, orfrom about 350 kb to about 400 kb; (e) comprises an exogenous nucleicacid sequence comprising a homologous or an orthologous nucleic acidsequence; (f) comprises a chimeric nucleic acid sequence comprising ahuman and a rat nucleic acid sequence; (g) ranges from about 5 kb toabout 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,from about 80 kb to about 100 kb, from about 100 kb to about 150 kb,from about 150 kb to about 200 kb, from about 200 kb to about 250 kb,from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, orfrom about 350 kb to about 400 kb; (h) comprises a conditional alleleflanked with site-specific recombinase target sequences; or, (i)comprises a reporter gene operably linked to a promoter active in a ratcell.

Further provided is a method for modifying a genomic locus of interestin a pluripotent rat cell via targeted genetic modification, wherein thegenomic locus of interest comprises (i) a first nucleic acid sequencethat is complementary to the 5′ rat homology arm; and (ii) a secondnucleic acid sequence that is complementary to the 3′ rat homology arm.In some such embodiments, the first and the second nucleic acid sequenceis separated by at least 5 kb. In some embodiments, the first and thesecond nucleic acid sequence is separated by at least 5 kb but less than3 Mb. In some such methods, the first and the second nucleic acidsequence is separated by at least 5 kb but less than 10 kb, at least 10kb but less than 20 kb, at least 20 kb but less than 40 kb, at least 40kb but less than 60 kb, at least 60 kb but less than 80 kb, at leastabout 80 kb but less than 100 kb, at least 100 kb but less than 150 kb,or at least 150 kb but less than 200 kb, at least about 200 kb but lessthan about 300 kb, at least about 300 kb but less than about 400 kb, atleast about 400 kb but less than about 500 kb, at least about 500 kb butless than about 1 Mb, at least about 1 Mb but less than about 1.5 Mb, atleast about 1.5 Mb but less than about 2 Mb, at least about 2 Mb butless than about 2.5 Mb, at least about 2.5 Mb but less than about 3 Mb,at least about 1 Mb but less than about 2 Mb, at least about 2 Mb butless than about 3 Mb.

In some embodiments, the introducing step further comprises introducinga second nucleic acid encoding a nuclease agent that promotes ahomologous recombination between the targeting construct and the genomiclocus of interest in the pluripotent rat cell. In some such embodiments,the nuclease agent comprises (a) a chimeric protein comprising a zincfinger-based DNA binding domain fused to a FokI endonuclease; or, (b) achimeric protein comprising a Transcription Activator-Like EffectorNuclease (TALEN) fused to a FokI endonuclease.

In some methods, the introducing step further comprises introducing intothe pluripotent rat cell: (i) a first expression construct comprising afirst promoter operably linked to a first nucleic acid sequence encodinga Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)-associated (Cas) protein, (ii) a second expression constructcomprising a second promoter operably linked to a second nucleic acidsequence encoding a genomic target sequence operably linked to a guideRNA (gRNA), wherein the genomic target sequence is immediately flankedon the 3′ end by a Protospacer Adjacent Motif (PAM) sequence. In oneembodiment, the genomic locus of interest comprises the nucleotidesequence of SEQ ID NO: 1. In one embodiment, the gRNA comprises a thirdnucleic acid sequence encoding a Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR) RNA (crRNA) and a trans-activating CRISPRRNA (tracrRNA). In another embodiment, the genome of the pluripotent ratcell comprises a target DNA region complementary to the genomic targetsequence. In some such methods, the Cas protein is Cas9. In some suchmethods the gRNA comprises (a) the chimeric RNA of the nucleic acidsequence of SEQ ID NO: 2; or, (b) the chimeric RNA of the nucleic acidsequence of SEQ ID NO: 3. In some such methods, the crRNA comprises thesequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. Insome such methods, the tracrRNA comprises the sequence set forth in SEQID NO: 7 or SEQ ID NO: 8.

Further provided is a rat genomic locus comprising (i) an insertion of ahomologous or orthologous human nucleic acid sequence; (ii) areplacement of an endogenous rat nucleic acid sequence with thehomologous or orthologous human nucleic acid sequence; or (iii) acombination thereof, wherein the rat genomic locus is capable of beingtransmitted through the germline. In some such rat genomic locus, thesize of the insertion or replacement is from about 5 kb to about 400 kb.In some such rat genomic locus, the size of the insertion or replacementis from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, fromabout 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kbto about 250 kb, from about 250 kb to about 300 kb, from about 300 kb toabout 350 kb, from about 350 kb to about 400 kb, from about 400 kb toabout 800 kb, from about 800 kb to 1 Mb, from about 1 Mb to about 1.5Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb, to about 2.5 Mb,from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb to about 3 Mb, atleast about 200 kb but less than about 300 kb, at least about 300 kb butless than about 400 kb, at least about 400 kb but less than about 500kb, at least about 500 kb but less than about 1 Mb, at least about 1 1Mb but less than about 2 Mb, at least about 2 Mb but less than about 3Mb.

Further provided is a method for making a humanized rat, comprising: (a)targeting a genomic locus of interest in a pluripotent rat cell with atargeting construct comprising a human insert nucleic acid to form agenetically modified pluripotent rat cell; (b) introducing thegenetically modified pluripotent rat cell into a host rat embryo; and(c) gestating the host rat embryo in a surrogate mother; wherein thesurrogate mother produces rat progeny comprising, a modified genomiclocus that comprises: (i) an insertion of a human nucleic acid sequence;(ii) a replacement of the rat nucleic acid sequence at the genomic locusof interest with a homologous or orthologous human nucleic acidsequence; (iii) a chimeric nucleic acid sequence comprising a human anda rat nucleic acid sequence; or (iv) a combination thereof, wherein themodified genomic locus is capable of being transmitted through thegermline.

In some such methods, the targeting construct is a large targetingvector (LTVEC), and the sum total of the 5′ and the 3′ homology arms ofthe LTVEC is at least 10 kb but less than 100 kb or the sum total of the5′ and the 3′ homology arms of the LTVEC is at least 10 kb but less than150 kb. In some such methods, the sum total of the 5′ and the 3′homology arms of the targeting construct is from about 10 kb to about 30kb, from about 20 kb to 40 kb, from about 40 kb to about 60 kb, fromabout 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about100 kb to about 120 kb, or from about 120 kb to about 150 kb. In somesuch methods, the human nucleic acid sequence is at least 5 kb but lessthan 400 kb. In some such methods, the human nucleic acid sequence is atleast 5 kb but less than 10 kb, at least 10 kb but less than 20 kb, atleast 20 kb but less than 40 kb, at least 40 kb but less than 60 kb, atleast 60 kb but less than 80 kb, at least about 80 kb but less than 100kb, at least 100 kb but less than 150 kb, at least 150 kb but less than200 kb, at least 200 kb but less than 250 kb, at least 250 kb but lessthan 300 kb, at least 300 kb but less than 350 kb, or at least 350 kbbut less than 400 kb. In other embodiments, the size of the sum total ofthe total of the 5′ and 3′ homology arms of the LTVEC is about 10 kb toabout 150 kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb,about 20 kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb toabout 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100 kb,about 30 kb to about 75 kb, about 40 kb to about 150 kb, about 40 kb toabout 100 kb, about 40 kb to about 75 kb, about 50 kb to about 150 kb,about 50 kb to about 100 kb, about 50 kb to about 75 kb, about 10 kb toabout 30 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb,about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb toabout 120 kb, or from about 120 kb to about 150 kb. In one embodiment,the size of the deletion is the same or similar to the size of the sumtotal of the 5′ and 3′ homology arms of the LTVEC.

In some methods for making a humanized rat, the pluripotent rat cell isa rat embryonic stem (ES) cell. In some such methods, the pluripotentrat cell is derived from a DA strain or an ACI strain. In some suchmethods, the pluripotent rat cell is characterized by expression of atleast one pluripotency marker comprises Dnmt3L, Eras, Err-beta, Fbxo15,Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2,Utf1, and/or a combination thereof. In some such methods, thepluripotent rat cell is characterized by one or more of the followingfeatures: (a) lack of expression of one or more pluripotency markerscomprising c-Myc, Ecat1, and/or Rexo1; (b) lack of expression of one ormore mesodermal markers comprising Brachyury and/or Bmpr2; (c) lack ofexpression of one or more endodermal markers comprising Gata6, Sox17,and/or Sox7; or (d) lack of expression of one or more neural markerscomprising Nestin and/or Pax6.

Further provided is a genetically modified rat comprising a humanizedgenomic locus, wherein the genetically modified rat comprises: (i) aninsertion of a homologous or orthologous human nucleic acid sequence;(ii) a replacement of a rat nucleic acid sequence with a homologous ororthologous human nucleic acid sequence at an endogenous genomic locuswith a homologous or orthologous human nucleic acid sequence; (iii) achimeric nucleic acid sequence comprising a human and a rat nucleic acidsequence; or, (iv) a combination thereof, wherein the humanized genomiclocus is capable of being transmitted through the germline. In some suchgenetically modified rats, the humanized genomic locus comprises achimeric nucleic acid sequence comprising a human and a rat nucleic acidsequence.

Methods for modifying a target genomic locus of a rat via bacterialhomologous recombination (BHR) are also provided and comprise:introducing into a prokaryotic cell a large targeting vector (LTVEC)comprising an insert nucleic acid flanked with a 5′ rat homology arm anda 3′ rat homology arm, wherein the prokaryotic cell comprises a ratnucleic acid and is capable of expressing a recombinase that mediatesthe BHR at the target locus, and wherein the sum total of the 5′ and 3′homology arms of the LTVEC is at least 10 kb but less than 100 kb or thesum total of the 5′ and the 3′ homology arms of the LTVEC is at least 10kb but less than 150 kb. In other embodiments, the size of the sum totalof the total of the 5′ and 3′ homology arms of the LTVEC is about 10 kbto about 150 kb, about 10 kb to about 100 kb, about 10 kb to about 75kb, about 20 kb to about 150 kb, about 20 kb to about 100 kb, about 20kb to about 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100kb, about 30 kb to about 75 kb, about 40 kb to about 150 kb, about 40 kbto about 100 kb, about 40 kb to about 75 kb, about 50 kb to about 150kb, about 50 kb to about 100 kb, or about 50 kb to about 75 kb, about 10kb to about 30 kb, about 20 kb to about 40 kb, about 40 kb to about 60kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100kb to about 120 kb, or from about 120 kb to about 150 kb. In oneembodiment, the size of the deletion is the same or similar to the sizeof the sum total of the 5′ and 3′ homology arms of the LTVEC.

In some such methods, the target locus of the rat nucleic acid comprisesa first nucleic acid sequence that is complementary to the 5′ homologyarm and a second nucleic acid sequence that is complementary to the 3′homology arm. In some such methods, the first and the second nucleicacid sequence is separated by at least 5 kb but less than 10 kb, atleast 10 kb but less than 20 kb, at least 20 kb but less than 40 kb, atleast 40 kb but less than 60 kb, at least 60 kb but less than 80 kb, atleast about 80 kb but less than 100 kb, at least 100 kb but less than150 kb, or at least 150 kb but less than 200 kb, at least about 200 kbbut less than about 300 kb, at least about 300 kb but less than about400 kb, at least about 400 kb but less than about 500 kb, at least about500 kb but less than about 1 Mb, at least about 1 1 Mb but less thanabout 2 Mb, at least about 2 Mb but less than about 3 Mb.

In some such methods, introducing the targeting vector into theprokaryotic cell leads to: (i) a deletion of an endogenous rat nucleicacid sequence from the target genomic locus; (ii) an addition of anexogenous nucleic acid sequence at the target genomic locus; (iii) areplacement of the endogenous rat nucleic acid sequence with theexogenous nucleic acid sequence at the target locus; or (iv) acombination thereof. In some such methods, the insert nucleic acidcomprises (a) a polynucleotide that is homologous or orthologous to therat nucleic acid sequence at the target genomic locus; or (b) aconditional allele flanked with site-specific recombination recognitionsequences.

Further provided is a host prokaryotic cell comprising a targetingvector comprising an insert nucleic acid flanked with a 5′ rat homologyarm and a 3′ rat homology arm, wherein the insert nucleic acid rangesfrom about 5 k to about 400 kb. In some host prokaryotic cells the sizeof the insert nucleic acid is from about 5 kb to about 10 kb, from about10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kbto about 60 kb, from about 60 kb to about 80 kb, from about 80 kb toabout 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, or from 350 kb to about400 kb. In some host prokaryotic cells, the prokaryotic cell comprises arecombinase gene operably linked to a constitutively active promoter oran inducible promoter.

Methods are also provided for modifying a genomic locus of interest in acell via targeted genetic modification comprising introducing into thecell

(a) a large targeting vector (LTVEC) comprising an insert nucleic acidflanked with a 5′ homology arm and a 3′ homology arm, wherein the sumtotal of the 5′ and 3′ homology arms of the LTVEC is at least 10 kb; and

(b) (i) a first expression construct comprising a first promoteroperably linked to a first nucleic acid sequence encoding a ClusteredRegularly Interspaced Short Palindromic Repeats (CRISPR)-associated(Cas) protein, (ii) a second expression construct comprising a secondpromoter operably linked to a second nucleic acid sequence encoding agenomic target sequence operably linked to a guide RNA (gRNA); andidentifying a genetically modified pluripotent cell comprising thetargeted genetic modification at the genomic locus of interest.

In one embodiment, the genomic locus of interest comprises thenucleotide sequence set forth in SEQ ID NO: 1, wherein the gRNAcomprises a third nucleic acid sequence encoding a Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) and atrans-activating CRISPR RNA (tracrRNA), and wherein the genome of thecell comprises a target DNA region complementary to the genomic targetsequence. In some such methods, the Cas protein is Cas9. In suchmethods, the cell can be a pluripotent cell (such as an embryonic stemcell) or a prokaryotic cell. In one embodiment, the pluripotent cell isfrom non-human animal, a non-human mammal, a rodent, a human, a rat, amouse, a hamster a rabbit, a pig, a bovine, a deer, a sheep, a goat, achicken, a cat, a dog, a ferret, a primate (e.g., marmoset, rhesusmonkey), domesticated mammal or an agricultural mammal or any otherorganism of interest. In another embodiment, the prokaryotic cell isfrom bacteria, such as, E. coli.

In other embodiments, the size of the sum total of the total of the 5′and 3′ homology arms of the LTVEC is about 10 kb to about 150 kb, about10 kb to about 100 kb, about 10 kb to about 75 kb, about 20 kb to about150 kb, about 20 kb to about 100 kb, about 20 kb to about 75 kb, about30 kb to about 150 kb, about 30 kb to about 100 kb, about 30 kb to about75 kb, about 40 kb to about 150 kb, about 40 kb to about 100 kb, about40 kb to about 75 kb, about 50 kb to about 150 kb, about 50 kb to about100 kb, or about 50 kb to about 75 kb, about 10 kb to about 30 kb, about20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about80 kb, about 80 kb to about 100 kb, about 100 kb to about 120 kb, orfrom about 120 kb to about 150 kb. In one embodiment, the size of thedeletion is the same or similar to the size of the sum total of the 5′and 3′ homology arms of the LTVEC.

In one embodiment, the pluripotent cell is a non-human pluripotent cell.In one embodiment, the non-human pluripotent cell is a mammalianpluripotent cell. In one embodiment, the mammalian pluripotent cell is arodent pluripotent cell. In one embodiment, the rodent pluripotent cellis a rat or mouse pluripotent cell. In one embodiment, the pluripotentcell is a human induced pluripotent stem (iPS) cell.

In one embodiment, the pluripotent cell is a non-human fertilized egg atthe single cell stage. In one embodiment, the non-human fertilized eggis a mammalian fertilized egg. In one embodiment, the mammalianfertilized egg is a rodent fertilized egg at the single cell stage. Inone embodiment, the mammalian fertilized egg is a rat or mousefertilized egg at the single cell stage.

Further provided is a rat or rat cell comprising a targeted geneticmodification in its genomic locus, wherein the genomic locus is anInterleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2locus, or a Rag2/Rag1 locus, wherein the targeted genetic modificationcomprises: (a) a deletion of an endogenous rat nucleic acid sequence atthe genomic locus; (b) an insertion of a homologous nucleic acid, anorthologous nucleic acid, or a chimeric nucleic acid comprising a humanand a rat nucleic acid sequence; or (c) a combination thereof. In such arat or rat cell, the targeted genetic modification is transmissiblethrough the germline of the rat or a rat propagated from the rat cell.

In some such rats or rat cells the deletion of the endogenous ratnucleic acid at the genomic locus is at least about 10 kb, or theinsertion of the exogenous nucleic acid sequence at the genomic locus isat least about 5 kb.

Further provided is a rat or rat cell, wherein (a) the targeted geneticmodification at the Interleukin-2 receptor gamma locus results in adecrease in or absence of Interleukin-2 receptor gamma protein activity;(b) the targeted genetic modification at the ApoE locus results in adecrease in or absence of ApoE protein activity; (c) the targetedgenetic modification at the Rag1 locus results in a decrease in orabsence of Rag1 protein activity; (d) the targeted genetic modificationat the Rag2 locus results in a decrease in or absence of Rag2 proteinactivity; or, (e) the targeted genetic modification at the Rag2/Rag1locus results in a decrease in or absence of Rag2 protein activity andRag1 activity.

In some embodiments, the targeted genetic modification of theInterleukin-2 receptor gamma locus comprises: (a) a deletion of theentire rat Interleukin-2 receptor gamma coding region or a portionthereof; (b) a replacement of the entire rat Interleukin-2 receptorgamma coding region or a portion thereof with a human Interleukin-2receptor gamma coding region or a portion thereof; (c) a replacement ofan ecto-domain of the rat Interleukin-2 receptor gamma coding regionwith the ecto-domain of a human Interleukin-2 receptor gamma; or, (d) atleast a 3 kb deletion of the Interleukin-2 receptor gamma locus. Inother such rats or rat cells the targeted genetic modification of theApoE locus comprises: (a) a deletion of the entire ApoE coding region ora portion thereof; or, (b) at least a 1.8 kb deletion of the ApoE locuscomprising the ApoE coding region.

Further provided is a rat or rat cell, wherein the targeted geneticmodification of the Rag2 locus comprises: (a) a deletion of the entireRag2 coding region or a portion thereof; or (b) at least a 5.7 kbdeletion of the Rag2 locus comprising the Rag2 coding region. In someembodiments, the targeted genetic modification of the Rag2/Rag1 locuscomprises: (a) a deletion of the entire Rag2 coding region or a portionthereof and a deletion of the entire Rag1 coding region or portionthereof; or, (b) a deletion of at least 16 kb of the Rag2/Rag1 locuscomprising the Rag2 coding region.

Further provided is a rat or rat cell, wherein the targeted geneticmodification comprises an insertion of an expression cassette comprisinga selective marker at the Interleukin-2 receptor gamma locus, the ApoElocus, the Rag1 locus, the Rag2 locus, or the Rag2/Rag1 locus. In somesuch rats or rat cells the expression cassette comprises a lacZ geneoperably linked to the endogenous promoter at the genomic locus and ahuman ubiquitin promoter operably linked to a selective marker.

Further provided is a rat or rat cell, wherein the targeted geneticmodification in the Interleukin-2 receptor gamma locus, the ApoE locus,the Rag1 locus, the Rag2 locus or the Rag2/Rag1 locus comprises theinsertion of a self-deleting selection cassette. In some such rats orrat cells, the self-deleting selection cassette comprises a selectivemarker gene operably linked to a promoter active in the rat cell and arecombinase gene operably linked to a male germ cell-specific promoter,wherein the self-deleting cassette is flanked by recombinationrecognition sites recognized by the recombinase. In some such rats orrat cells, the male germ cell-specific promoter is a Protamine-1promoter; the recombinase gene encodes Cre, and the recombinationrecognition sites are loxP sites. In one embodiment, the Protamine-1promoter is a mouse or a rat Protamine-1 promoter.

Further provided is a rat or rat cell, wherein the insertion of theexogenous nucleic acid sequence at the genomic locus comprises areporter nucleic acid operably linked to an endogenous Interleukin-2receptor gamma promoter, an endogenous ApoE promoter, an endogenous Rag1promoter, or an endogenous Rag2 promoter. In some such rats or ratcells, the reporter nucleic acid encodes a reporter comprisingβ-galactosidase, mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed,mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein(EYFP), Emerald, enhanced green fluorescent protein (EGFP), CyPet, cyanfluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkalinephosphatase, or a combination thereof.

Further provided is a rat cell, wherein the rat cell is a pluripotentrat cell or a rat embryonic stem (ES) cell. In some such rat cells, thepluripotent rat cell or the rat embryonic stem (ES) cell (a) is derivedfrom a DA strain or an ACI strain; (b) is characterized by expression ofat least one pluripotency marker comprising Dnmt3L, Eras, Err-beta,Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15,Sox2, Utf1, or a combination thereof; or (c) is characterized by one ormore of the following characteristics: (i) lack of expression of one ormore pluripotency markers comprising c-Myc, Ecat1, and Rexo1; (ii) lackof expression of mesodermal markers comprising Brachyury and Bmpr2;(iii) lack of expression of one or more endodermal markers comprisingGata6, Sox17 and Sox7; or (iv) lack of expression of one or more neuralmarkers comprising Nestin and Pax6.

Further provided is a method for modifying a target genomic locus in anInterleukin-2 receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2locus or a Rag2/Rag1 locus in a pluripotent rat cell, the methodcomprising: (a) introducing into the pluripotent rat cell a targetingvector comprising an insert nucleic acid flanked with 5′ and 3′ rathomology arms homologous to the target genomic locus; and (b)identifying a genetically modified pluripotent rat cell comprising atargeted genetic modification at the target genomic locus, wherein thetargeted genetic modification is capable of being transmitted throughthe germline of a rat propagated from the pluripotent rat cell. In somesuch methods, the targeting vector is a large targeting vector (LTVEC),wherein the sum total of the 5′ and the 3′ rat homology arms is at leastabout 10 kb. In some embodiments, the sum total of the 5′ and the 3′ rathomology arms is at least 10 kb but less than 150 kb. In someembodiments, the sum total of the 5′ and the 3′ rat homology arms is atleast about 10 kb but less than about 100 kb. In some embodiments,introducing the targeting vector into the pluripotent rat cell leads to:(i) a deletion of an endogenous rat nucleic acid sequence at the targetgenomic locus; (ii) an insertion of an exogenous nucleic acid sequenceat the target genomic locus; or (iii) a combination thereof.

In some embodiments, the deletion of the endogenous rat nucleic acid atthe genomic locus is at least about 10 kb; the deletion of an endogenousrat nucleic acid sequence at the genomic locus ranges from about 5 kb toabout 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, orfrom about 150 kb to about 200 kb, from about 200 kb to about 300 kb,from about 300 kb to about 400 kb, from about 400 kb to about 500 kb,from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, fromabout 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or fromabout 2.5 Mb to about 3 Mb; the insertion of an exogenous nucleic acidsequence at the genomic locus is at least about 5 kb; or the insertionof an exogenous nucleic acid sequence ranges from about 5 kb to about 10kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, fromabout 80 kb to about 100 kb, from about 100 kb to about 150 kb, fromabout 150 kb to about 200 kb, from about 200 kb to about 250 kb, fromabout 250 kb to about 300 kb, from about 300 kb to about 350 kb, or fromabout 350 kb to about 400 kb.

In some embodiments, (a) the targeted genetic modification at theInterleukin-2 receptor gamma locus results in a decrease in or absenceof Interleukin-2 receptor gamma protein activity; (b) the targetedgenetic modification at the ApoE locus results in a decrease in orabsence of ApoE protein activity; (c) the targeted genetic modificationat the Rag1 locus results in a decrease in or absence of Rag1 proteinactivity; (d) the targeted genetic modification at the Rag2 locusresults in a decrease in or absence of Rag2 protein activity; or, (e)the targeted genetic modification at the Rag2/Rag1 locus results in adecrease in or absence of Rag2 protein activity and Rag1 proteinactivity.

In some embodiments, the targeted genetic modification at theInterleukin-2 receptor gamma locus comprises (a) a deletion of theentire rat Interleukin-2 receptor gamma coding region or a portionthereof; (b) a replacement of the entire rat Interleukin-2 receptorgamma coding region or a portion thereof with a human Interleukin-2receptor gamma coding region or a portion thereof; (c) a replacement ofan ecto-domain of the rat Interleukin-2 receptor gamma coding regionwith the ecto-domain of a human Interleukin-2 receptor gamma; or, (d) atleast a 3 kb deletion of the Interleukin-2 receptor gamma locuscomprising the Interleukin-2 receptor gamma coding region.

In some embodiments, the targeted genetic modification at the ApoE locuscomprises: (a) a deletion of the entire ApoE coding region or a portionthereof; or, (b) at least a 1.8 kb deletion of the ApoE locus comprisingthe ApoE coding region.

In some embodiments, the targeted genetic modification at the Rag2 locuscomprises: (a) a deletion of the entire Rag2 coding region or a portionthereof; or, (b) at least a 5.7 kb deletion of the Rag2 locus comprisingthe Rag2 coding region. In other methods, the targeted geneticmodification of the Rag1/Rag2 locus comprises: (a) a deletion of theentire Rag2 coding region or a portion thereof and a deletion of theentire Rag1 coding region or portion thereof; or, (b) a deletion of atleast 16 kb of the Rag2/Rag1 locus comprising the Rag2 and Rag1 codingregions.

In some such embodiments for modifying a target genomic locus, theinsert nucleic acid comprises an expression cassette comprising apolynucleotide encoding a selective marker. In some such embodiments,the expression cassette comprises a lacZ gene operably linked to anendogenous promoter at the genomic locus and a human ubiquitin promoteroperably linked to a selective marker gene.

In some embodiments, the insert nucleic acid comprises a self-deletingselection cassette. In some such embodiments, the self-deletingselection cassette comprises a selective marker operably linked to apromoter active in the rat pluripotent cell and a polynucleotideencoding a recombinase operably linked to a male germ cell-specificpromoter, wherein the self-deleting cassette is flanked by recombinationrecognition sites recognized by the recombinase. In some suchembodiments, the male germ cell-specific promoter is a Protamine-1promoter; or, the recombinase gene encodes Cre and the recombinationrecognition sites are loxP sites. In some embodiments, the Protamine-1promoter is a mouse or a rat Protamine-1 promoter.

In other methods, the insertion of the exogenous nucleic acid sequenceat the genomic locus comprises a reporter nucleic acid sequence operablylinked to the endogenous Interleukin-2 receptor gamma promoter, theendogenous ApoE promoter, the endogenous Rag1 promoter, or theendogenous Rag2 promoter. In some such embodiments, the reporter nucleicacid sequence encodes a reporter comprising β-galactosidase, mPlum,mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine,Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald,enhanced green fluorescent protein (EGFP), CyPet, cyan fluorescentprotein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase,or a combination thereof.

In some embodiments for modifying a target genomic locus, thepluripotent rat cell is a rat embryonic stem (ES) cell. In some suchembodiments, the pluripotent rat cell (a) is derived from a DA strain oran ACI strain; (b) is characterized by expression of a pluripotencymarker comprising Oct-4, Sox-2, alkaline phosphatase, or a combinationthereof; or, (c) is characterized by one or more of the followingcharacteristics: (i) lack of expression of one or more pluripotencymarkers comprising c-Myc, Ecat1, and Rexo1; (ii) lack of expression ofmesodermal markers comprising Brachyury and Bmpr2; (iii) lack ofexpression of one or more endodermal markers comprising Gata6, Sox17 andSox7; or (iv) lack of expression of one or more neural markerscomprising Nestin and Pax6.

In some embodiments, the method further comprises identifying thetargeted genetic modification at the target genomic locus, wherein theidentification step employs a quantitative assay for assessing amodification of allele (MOA) at the target genomic locus.

In some embodiments, the introducing step further comprises introducinga second nucleic acid encoding a nuclease agent that promotes ahomologous recombination between the targeting vector and the targetgenomic locus in the pluripotent rat cell. In some such embodiments, thenuclease agent comprises a chimeric protein comprising a zincfinger-based DNA binding domain fused to a FokI endonuclease. Some suchmethods result in bi-allelic modification of the target genomic locus.

In some embodiments, the introducing step of the method furthercomprises introducing into the pluripotent rat cell: a first expressionconstruct comprising a first promoter operably linked to a first nucleicacid sequence encoding a Clustered Regularly Interspaced ShortPalindromic Repeats (CRISPR)-associated (Cas) protein, and a secondexpression construct comprising a second promoter operably linked to asecond nucleic acid sequence encoding a genomic target sequence operablylinked to a guide RNA (gRNA), wherein the genomic target sequence isimmediately flanked on the 3′ end by a Protospacer Adjacent Motif (PAM)sequence. In one embodiment, the genomic target sequence comprises thenucleotide sequence set forth in SEQ ID NO: 1. In one embodiment thegRNA comprises a third nucleic acid sequence encoding a ClusteredRegularly Interspaced Short Palindromic Repeats (CRISPR) RNA (crRNA) anda trans-activating CRISPR RNA (tracrRNA). In some such embodiments, theCas protein is Cas9. In some such embodiments, (a) the gRNA is thechimeric RNA of the nucleic acid sequence set forth in SEQ ID NO: 2; (b)the gRNA is the chimeric RNA of the nucleic acid sequence set forth inSEQ ID NO: 3; (c) the crRNA comprises a sequence set forth in SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6; or, (d) the tracrRNA comprises thesequence set forth in SEQ ID NO: 7 and/or SEQ ID NO: 8.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts rat ESCs, which grow as compact spherical colonies thatroutinely detach and float in the dish.

FIGS. 2A through 2D depict various pluripotency markers expressed by ratESCs: FIG. 2A depicts Oct-4 (green); FIG. 2B depicts Sox-2 (red); FIG.2C depicts DAPI (blue); FIG. 2D depicts an overlay of pluripotencymarkers expressed by rESCs.

FIG. 3 depicts that the rat ESCs express light levels of alkalinephosphatase (a pluripotency marker)(left), and the karyotype for lineDA.2B is 42X,Y (right). Karyotyping was done because rat ESCs oftenbecome tetraploid; lines were thus pre-screened by counting metaphasechromosome spreads, and lines with mostly normal counts were thenformally karyotyped.

FIGS. 4A-4B provide photographs showing the analysis of the chromosomenumber of the ACI.G1 rat ES cell line.

FIGS. 5A-5B provide photographs showing the analysis of the chromosomenumber of the DA.2B rat ES cell line.

FIGS. 6A-6B provide photographs showing the analysis of the chromosomenumber of the DA.C2 rat ES cell line.

FIG. 7 depicts a closer view of a rat ESC of FIG. 1.

FIG. 8 depicts production of chimeras by blastocyst injection andtransmission of the rat ESC genome through the germline; chimerasproduced by blastocyst injection using parental ACI.G1 rat ESCs; highpercentage chimeras usually have albino snouts.

FIG. 9 depicts F1 agouti pups with albino littermates, sired by ACI/SDchimera labeled with an asterisk (*) in FIG. 8.

FIG. 10 provides a schematic of the rat ApoE locus and denotes with greybars the cutting site for zinc finger nucleases (ZFN1 and ZFN2). Thegenomic regions corresponding to the 5′ and 3′ homology arms (5 kb and5.4 kb, respectively) are denoted by the dark grey boxes. Exon 1 of theApoE gene is non-coding and is shown as an open box closest to the 5′homology arm. The three introns of the ApoE gene are denoted as lines.Exons 2 and 3 comprise coding regions and are shown as stippled greyboxes. Exon 4 contains both coding and non-coding sequences as denotedby the stippled grey shading and the open box.

FIG. 11 provides a summary of the ApoE targeting efficiency whenperformed in the presence of zinc finger nucleases (ZFN1 or ZFN2)

FIGS. 12A-12C depict targeting of the rat Rosa 26 locus, which liesbetween the Setd5 and Thumpd3 genes as in mouse, with the same spacing.FIG. 12A shows the structure of the mouse Rosa 26 locus. Mouse Rosa26transcripts consist of 2 or 3 exons. FIG. 12B depicts the structure ofthe rat Rosa26 locus; the rat locus contains a second exon 1 (Ex1b) inaddition to the homologous exon to mouse exon1 (Ex1a); no third exon hasbeen identified in rat. FIG. 12C depicts a targeted rat Rosa26 allele;homology arms of 5 kb each were cloned by PCR using genomic DNA from DArESC; the targeted allele contains a Splicing Acceptor (SA)-lacZ-hUB-neocassette replacing a 117 bp deletion in the rat Rosa26 intron.

FIG. 13A depicts a control brain of a 14-week-old wild type rat, whichwas stained with X-gal. The control brain showed a low level ofbackground staining for LacZ (dorsal view).

FIG. 13B depicts LacZ expression in the brain of an rRosa26 heterozygousrat (14-week old). The lacZ reporter was expressed ubiquitouslythroughout the brain of the rRosa26 heterozygote.

FIG. 13C depicts a control heart and thymus (inset) of a 14-week-oldwild type rat, which were treated with X-gal. The control heart andthymus showed a low level of background staining for LacZ.

FIG. 13D depicts LacZ expression in the heart and thymus (inset) of a14-week-old rRosa26 heterozygous rat. The lacZ reporter was expressedubiquitously throughout the heart and thymus of the rROSA26heterozygote.

FIG. 13E depicts a control lung of a 14-week-old wild type rat, whichwas treated with X-gal. The control lung showed a low level ofbackground staining for LacZ.

FIG. 13F depicts LacZ expression in the lung of a 14-week-old rRosa26heterozygote rat. The lacZ reporter was expressed ubiquitouslythroughout the lung of the rRosa26 heterozygote.

FIGS. 13G and 13H depict LacZ expression in E12.5 rat embryos. Incontrast to the wild-type control embryo (FIG. 13H), which shows a lowlevel of background LacZ staining, the rRosa26 heterozygous embryoexhibited ubiquitous expression of the LacZ reporter throughout theembryo (FIG. 13G).

FIGS. 13I and 13J depict LacZ expression in E14.5 rat embryos. Incontrast to the wild-type control embryo (FIG. 13J), which shows a lowlevel of background LacZ staining, the rRosa26 heterozygous rat embryoexhibited ubiquitous expression of the LacZ reporter throughout theembryo (FIG. 13I).

FIG. 14 illustrates a homologous or non-homologous recombination eventthat occurs inside a rat ES cell following an electroporation of atargeting vector comprising a selection cassette (lacZ-neo cassette).

FIG. 15 illustrates the mechanism by which genome-editing endonucleases(e.g., ZFNs and TALENs) introduce a double strand break (DSB) in atarget genomic sequence and activate non-homologous end-joining (NHEJ)in an ES cell.

FIG. 16 illustrates a gene targeting technique that utilizes ZFN/TALENsto improve the efficiency of homologous recombination of a targetingvector. DSB represents double strand break.

FIG. 17 provides a summary of the chimera production and germlinetransmission of the modified rat ApoE locus. The targeted modificationwas assisted by zinc finger nucleases.

FIG. 18 provides a schematic of the IL2r-γ targeting event incombination with zinc finger nucleases that target ZFN U and ZFN D. ZFNcut sites are noted in the figure.

FIG. 19 provides the targeting efficiency when targeting IL2r-γ incombination with the CRISPR/Cas9 system.

FIG. 20 provides a schematic of the rat ApoE locus and a targetingplasmid. The upper schematic shows the genomic structure of the rat ApoElocus and the genomic regions corresponding to the 5′ and 3′ homologyarms (5 kb and 5.4 kb respectively; dark grey boxes). Exon 1 of the ApoEgene is non-coding and is shown as an open box closest to the 5′homology arm. The three introns of the ApoE gene are denoted as lines.Exons 2 and 3 comprise coding regions and are shown as stippled greyboxes. Exon 4 contains both coding and non-coding sequences as denotedby the stippled grey shading and the open box. The lower panel shows thetargeting plasmid. The 5′ and 3′ homology arms (5 kb and 5.4 kb,respectively) are denoted by the dark grey boxes. The targeting vectorcomprises a reporter gene (lacZ) and a self-deleting cassette flanked byloxP sites (open arrows). The self-deleting cassette comprises a mousePrm1 promoter operably linked to the Crei gene and a drug selectioncassette comprising a human ubiquitin promoter operably linked to aneomycin resistance gene.

FIG. 21 provides a schematic for targeting the ApoE locus in rat EScells using zinc-finger nucleases and a targeting vector comprising areporter gene (LacZ) and a self-deleting cassette comprising a mousePrm1 promoter operably linked to the Crei gene and a drug selectioncassette comprising a human ubiquitin promoter operably linked to aneomycin resistance gene.

FIG. 22 provides a schematic of the rat ApoE locus and a large targetingvector (LTVEC). The upper panel shows the genomic organization of therat ApoE locus and the genomic regions corresponding to the 5′ and 3′homology arms (45 kb and 23 kb, respectively; the dark grey boxes). Exon1 of ApoE is non-coding and is shown as an open box closet to the 5′homology arm. The three introns of the ApoE gene are denoted as linesand exons 2 and 3 comprise coding regions and are shown as stippled greyboxes. Exon 4 contains both coding and non-coding sequences as denotedby the stippled grey shading and the open box. The lower panel shows theLTVEC for modifying the rat ApoE locus. The 5′ and 3′ homology arms (45kb and 23 kb, respectively) are denoted by the dark grey boxes. TheLTVEC comprises a reporter gene (lacZ) and a self-deleting cassetteflanked by loxP sites (open arrows), which comprises a mouse Prm1promoter operably linked to the Crei gene and a drug selection cassettecomprising a human ubiquitin promoter operably linked to a neomycinresistance gene.

FIG. 23 provides a schematic of the rat ApoE locus and denotes with greybars the cutting sites for zinc finger nucleases (ZFN1 and ZFN2) usedtogether with the large targeting vector (LTVEC) to enhance homologousrecombination between the targeting vector and the target cognatechromosomal region.

FIG. 24 depicts the rat IL2r-γ locus that has been disrupted by a 3.2 kbdeletion and the insertion of a reporter gene (eGFP) and a self-deletingcassette comprising a drug selection cassette (hUb-neo) and the Creigene operably linked to a mouse Prm1 promoter.

FIG. 25 provides a summary of the germ-line transmitting, targetable ratembryonic stem cell lines.

FIG. 26 provides another depiction of the rat IL2r-γ locus that has beendisrupted by a 3.2 kb deletion and the insertion of a reporter gene(eGFP) and a self-deleting cassette comprising the Crei gene operablylinked to a mouse Prm1 promoter and a drug selection cassette (hUb-Neo).

FIG. 27 provides a schematic of the rat Rag2 locus and a large targetingvector (LTVEC) for modifying the rat Rag2 locus. The upper panel showsthe genomic organization of the rat Rag2 locus and the cognate genomicregions corresponding to the 5′ and 3′ homology arms (48 kb and 15 kb,respectively; dark grey boxes). Rag2 comprises single exon denoted bythe stippled grey shading. The lower panel is the LTVEC. The 5′ and 3′homology arms (48 kb and 15 kb, respectively) are denoted by the darkgrey boxes. The LTVEC comprises a reporter gene (lacZ) and aself-deleting cassette flanked by loxP sites (open arrows) that containsa rat Prm1 promoter operably linked to the Crei gene and a drugselection cassette containing a human ubiquitin promoter operably linkedto a neomycin resistance gene.

FIG. 28 provides the genomic structure of the rat Rag1/Rag2 locus andthe genomic regions deleted by either Rag2 targeting (Rag2 deletion) orRag2/Rag1 double targeting (Rag2/Rag1 deletion).

FIG. 29 provides a schematic of the rat Rag2 and Rag1 loci and a largetargeting vector (LTVEC) used for modifying the loci. The upper panelshows the genomic organization of the Rag1 and Rag2 loci and the cognategenomic regions corresponding to the 5′ and 3′ homology arms (48 kb and84 kb, respectively; dark grey boxes). Rag2 and Rag1 each comprise asingle exon denoted by the stippled grey shading. The lower panel is theLTVEC. The 5′ and 3′ homology arms (48 kb and 84 kb, respectively) aredenoted by the dark grey boxes. The LTVEC comprises a reporter gene(lacZ) and a self-deleting cassette flanked by loxP sites (open arrows),which comprises a rat Prm1 promoter operably linked to the Crei gene anda drug selection cassette comprising a human ubiquitin promoter operablylinked to a neomycin resistance gene.

FIG. 30 shows that Il2rg−/y PBMC do not express mature lymphocytemarkers. GFP-positive lymphocytes were detected in peripheral blood in 2of the 3 chimeras.

FIG. 31 provides a schematic of the rat IL-2rg locus and a targetingplasmid for the full humanization of the rat IL-2rg locus. The upperpanel shows the genomic organization of the rat IL-2rg locus and thecognate genomic regions corresponding to the 5′ and 3′ homology arms(4.4 kb and 5.0 kb, respectively; dark grey boxes). The lower panel isthe targeting plasmid. The 5′ and 3′ homology arms (4.4 kb and 5.0 kb,respectively) are denoted by the dark grey boxes. The targeting plasmidcomprises the human IL-2rg genomic region, a reporter gene (GFP) and aself-deleting cassette flanked by loxP sites (open arrows) that containsa mouse Prm1 promoter operably linked to the Crei gene and a drugselection cassette containing a human ubiquitin promoter operably linkedto a neomycin resistance gene.

FIG. 32 provides a schematic of the rat IL-2rg locus and a targetingplasmid for the ecto-domain humanization of the rat IL-2rg locus. Theupper panel shows the genomic organization of the rat IL-2rg locus andthe cognate genomic regions corresponding to the 5′ and 3′ homology arms(4.4 kb and 5.0 kb, respectively; dark grey boxes). The lower panel isthe targeting plasmid. The 5′ and 3′ homology arms (4.4 kb and 5.0 kb,respectively) are denoted by the dark grey boxes. The targeting plasmidcomprises the human ecto-domain of the IL-2Rg genomic region, a reportergene (GFP) and a self-deleting cassette flanked by loxP sites (openarrows) that contains a mouse Prm1 promoter operably linked to the Creigene and a drug selection cassette a human ubiquitin promoter operablylinked to a neomycin resistance gene.

FIG. 33 provides a sequence alignment of the human IL-2rg protein (SEQID NO: 20; NP 000197.1); the rat IL-2rg protein (SEQ ID NO: 21; NP543165.1); and the chimeric IL-2rg protein (SEQ ID NO: 22) comprisingthe human ecto-domain of IL-2rg fused to the remainder of the rat IL-2rgprotein. The junction between the human and rat IL-2rg is noted by thevertical line.

DETAILED DESCRIPTION OF THE INVENTION Glossary

The term “embryonic stem cell” or “ES cell” as used herein includes anembryo-derived totipotent or pluripotent cell that is capable ofcontributing to any tissue of the developing embryo upon introductioninto an embryo. The term “pluripotent cell” as used herein includes anundifferentiated cell that possesses the ability to develop into morethan one differentiated cell types.

The term “homologous nucleic acid” as used herein includes a nucleicacid sequence that is either identical or substantially similar to aknown reference sequence. In one embodiment, the term “homologousnucleic acid” is used to characterize a sequence having amino acidsequence that is at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or even 100% identical to a known reference sequence.

The term “orthologous nucleic acid” as used herein includes a nucleicacid sequence from one species that is functionally equivalent to aknown reference sequence in another species.

The term “large targeting vector” or “LTVEC” as used herein includeslarge targeting vectors for eukaryotic cells that are derived fromfragments of cloned genomic DNA larger than those typically used byother approaches intended to perform homologous gene targeting ineukaryotic cells. Examples of LTVEC, include, but are not limited to,bacterial homologous chromosome (BAC) and yeast artificial chromosome(YAC).

The term “modification of allele” (MOA) as used herein includes themodification of the exact DNA sequence of one allele of a gene(s) orchromosomal locus (loci) in a genome. Examples of “modification ofallele (MOA)” as described herein includes, but is not limited to,deletions, substitutions, or insertions of as little as a singlenucleotide or deletions of many kilobases spanning a gene(s) orchromosomal locus (loci) of interest, as well as any and all possiblemodifications between these two extremes.

The term “recombination site” as used herein includes a nucleotidesequence that is recognized by a site-specific recombinase and that canserve as a substrate for a recombination event.

“Serial” genetic modifications include two or more modifications to,e.g., a rat ES cell, conducted independently. For example, a firstmodification is made to a rat ES cell genome employing a suitable firstnucleic acid construct. The first modification may be achieved byelectroporation, or any other method known in the art. Then a secondmodification is made to the same rat ES cell genome employing a suitablesecond nucleic acid construct. The second modification may be achievedby a second electroporation, or any other method known in the art. Invarious embodiments, following the first and the second geneticmodifications of the same rat ES cell, a third, a fourth, a fifth, asixth, and so on, serial genetic modifications (one following another)may be achieved using, e.g., serial electroporation or any othersuitable method (serially) known in the art.

The term “site-specific recombinase” as used herein includes a group ofenzymes that can facilitate recombination between “recombination sites”where the two recombination sites are physically separated within asingle nucleic acid molecule or on separate nucleic acid molecules.Examples of “site-specific recombinase” include, but are not limited to,Cre, Flp, and Dre recombinases.

The term “germline” in reference to a nucleic acid sequence includes anucleic acid sequence that can be passed to progeny.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. Heavy chain variable domains are encoded by variable regionnucleotide sequence, which generally comprises V_(H), D_(H), and J_(H)segments derived from a repertoire of V_(H), D_(H), and J_(H) segmentspresent in the germline. Sequences, locations and nomenclature for V, D,and J heavy chain segments for various organisms can be found in IMGTdatabase, which is accessible via the internet on the world wide web(www) at the URL “imgt.org.”

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappa(κ) and lambda (λ) light chains and a VpreB, as well as surrogate lightchains. Light chain variable domains typically include three light chainCDRs and four framework (FR) regions, unless otherwise specified.Generally, a full-length light chain includes, from amino terminus tocarboxyl terminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region aminoacid sequence. Light chain variable domains are encoded by the lightchain variable region nucleotide sequence, which generally compriseslight chain V_(L) and light chain J_(L) gene segments, derived from arepertoire of light chain V and J gene segments present in the germline.Sequences, locations and nomenclature for light chain V and J genesegments for various organisms can be found in IMGT database, which isaccessible via the internet on the world wide web (www) at the URL“imgt.org.” Light chains include those, e.g., that do not selectivelybind either a first or a second epitope selectively bound by theepitope-binding protein in which they appear. Light chains also includethose that bind and recognize, or assist the heavy chain with bindingand recognizing, one or more epitopes selectively bound by theepitope-binding protein in which they appear.

The phrase “operably linked” comprises a relationship wherein thecomponents operably linked function in their intended manner. In oneinstance, a nucleic acid sequence encoding a protein may be operablylinked to regulatory sequences (e.g., promoter, enhancer, silencersequence, etc.) so as to retain proper transcriptional regulation. Inone instance, a nucleic acid sequence of an immunoglobulin variableregion (or V(D)J segments) may be operably linked to a nucleic acidsequence of an immunoglobulin constant region so as to allow properrecombination between the sequences into an immunoglobulin heavy orlight chain sequence.

1. Target Locus Comprising a Rat Nucleic Acid

Various methods and compositions are provided, which allow for theintegration of at least one insert nucleic acid at a target locus. Asused herein, a “genomic locus of interest” comprises any segment orregion of DNA within the genome that one desires to integrate an insertnucleic acid. The terms “genomic locus of interest” and “target genomiclocus of interest” can be used interchangeable. The genomic locus ofinterest can be native to the cell, or alternatively can comprise aheterologous or exogenous segment of DNA that was integrated into thegenome of the cell. Such heterologous or exogenous segments of DNA caninclude transgenes, expression cassettes, polynucleotide encodingselection makers, or heterologous or exogenous regions of genomic DNA.The term “locus” is a defined herein as a segment of DNA within thegenomic DNA. Genetic modifications as described herein can include oneor more deletions from a locus of interest, additions to a locus ofinterest, replacement of a locus of interest, and/or any combinationthereof. The locus of interest can comprise coding regions or non-codingregulatory regions.

The genomic locus of interest can further comprise any component of atargeted integration system including, for example, a recognition site,a selection marker, a previously integrated insert nucleic acid,polynucleotides encoding nuclease agents, promoters, etc. Alternatively,the genomic locus of interest can be located within a yeast artificialchromosome (YAC), bacterial artificial chromosome (BAC), a humanartificial chromosome, or any other engineered genomic region containedin an appropriate host cell. In various embodiments, the targeted locuscan comprise native, heterologous, or exogenous nucleic acid sequencefrom a prokaryote, a eukaryote, yeast, bacteria, a non-human mammal, anon-human cell, a rodent, a human, a rat, a mouse, a hamster, a rabbit,a pig, a bovine, a deer, a sheep, a goat, a chicken, a cat, a dog, aferret, a primate (e.g., marmoset, rhesus monkey), domesticated mammalor an agricultural mammal or any other organism of interest or acombination thereof.

In specific embodiments, the genomic locus of interest comprises atarget locus of a “rat nucleic acid”. Such a region comprises a nucleicacid from a rat that is integrated within the genome of a cell.

Non-limiting examples of the target locus include a genomic locus thatencodes a protein expressed in a B cell, a genomic locus that expressesa polypeptide in an immature B cell, a genomic locus that expresses apolypeptide in a mature B cell, an immunoglobulin (Ig) loci, or a T cellreceptor loci, including, for example, a T cell receptor alpha locus.Additional examples of target genomic locus include an FcER1a locus, aTLR4 locus, a PRLR locus, a Notch4 locus, an Accn2 locus, an Adamts5locus, a TRPA1 locus, FolH1 locus, an LRP5 locus, an IL2 receptor locus,including, for example, an IL2 Receptor gamma (IL2Rg) locus, an ApoElocus, a Rag1 locus, a Rag2 locus, a Rag1/Rag2 locus, and an ERBB4locus. Any such target locus can be from a rat.

In one embodiment, the target locus encodes a mammalian immunoglobulinheavy chain variable region amino acid sequence. In one embodiment, thetarget locus encodes a rat immunoglobulin heavy chain variable regionamino acid sequence. In one embodiment, the target locus comprises agenomic DNA sequence comprising an unrearranged rat, mouse, or humanimmunoglobulin heavy chain variable region nucleic acid sequenceoperably linked to an immunoglobulin heavy chain constant region nucleicacid sequence. In one embodiment, the immunoglobulin heavy chainconstant region nucleic acid sequence is a rat, mouse, or humanimmunoglobulin heavy chain constant region nucleic acid sequenceselected from a CH1, a hinge, a CH2, a CH3, and a combination thereof.In one embodiment, the heavy chain constant region nucleic acid sequencecomprises a CH1-hinge-CH2-CH3. In one embodiment, the target locuscomprises a rearranged rat, mouse, or human immunoglobulin heavy chainvariable region nucleic acid sequence operably linked to animmunoglobulin heavy chain constant region nucleic acid sequence. In oneembodiment, the immunoglobulin heavy chain constant region nucleic acidsequence is a rat, mouse, or human immunoglobulin heavy chain constantregion nucleic acid sequence selected from a CH1, a hinge, a CH2, a CH3,and a combination thereof. In one embodiment, the heavy chain constantregion nucleic acid sequence comprises a CH1-hinge-CH2-CH3.

In one embodiment, the target locus comprises a genomic DNA sequencethat encodes a mammalian immunoglobulin light chain variable regionamino acid sequence. In one embodiment, the genomic DNA sequencecomprises an unrearranged mammalian λ and/or κ light chain variableregion nucleic acid sequence.

In one embodiment, the genomic DNA sequence comprises a rearrangedmammalian λ and/or κ light chain variable region nucleic acid sequence.In one embodiment, the unrearranged λ or κ light chain variable regionnucleic acid sequence is operably linked to a mammalian immunoglobulinlight chain constant region nucleic acid sequence selected from a λlight chain constant region nucleic acid sequence and a κ light chainconstant region nucleic acid sequence. In one embodiment, the mammalianimmunoglobulin light chain constant region nucleic acid sequence is arat immunoglobulin light chain constant region nucleic acid sequence. Inone embodiment, the mammalian immunoglobulin light chain constant regionnucleic acid sequence is a mouse immunoglobulin light chain constantregion nucleic acid sequence. In one embodiment, the mammalianimmunoglobulin light chain constant region nucleic acid sequence is ahuman immunoglobulin light chain constant region nucleic acid sequence.

As used herein, a rat ApoE locus, a rat interleukin-2 receptor gamma(Il-2rg) locus, a rat Rag2 locus, a rat Rag1 locus and/or a ratRag2/Rag1 locus comprise the respective regions of the rat genome inwhich each of these genes or gene combinations are located. Modifyingany one of the rat ApoE locus, the rat interleukin-2 receptor gammalocus, the rat Rag2 locus, the rat Rag1 locus and/or the combined ratRag2/Rag1 locus can comprise any desired alteration to the given locus.Non-limiting examples of modification to the given rat locus arediscussed in further detail herein.

For example, in specific embodiments, one or more of the rat ApoE locus,the rat interleukin-2 receptor gamma locus, the Rag2 locus, and/or theRag2/Rag1 locus is modified such that the activity and/or level of theencoded ApoE protein or the interleukin-2 receptor gamma protein or theRag1 protein or the Rag2 protein or a combination of the Rag1 and Rag2proteins are decreased. In other embodiments, the activity of the ApoEprotein, the interleukin-2 receptor gamma protein, the Rag1 protein, orthe Rag2 protein, or a combination of the Rag1 and Rag2 proteins isabsent.

By “decreased” is intended any decrease in the level or activity of thegene/protein encoded at the locus of interest. For example, a decreasein activity can comprise either (1) a statistically significant decreasein the overall level or activity of a given protein (i.e., ApoE,interleukin-2 receptor gamma, Rag2, Rag2 or a combination of Rag1 andRag2) including, for example, a decreased level or activity of 0.5%, 1%,5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120% or greaterwhen compared to an appropriate control. Methods to assay for a decreasein the concentration and/or the activity of anyone of ApoE,interleukin-2 receptor gamma, Rag1 and Rag2 are known in the art.

In other embodiments, one or more of the rat ApoE locus, the ratinterleukin-2 receptor gamma locus, the rat Rag2 locus, the rat Rag1locus and/or rat Rag2/Rag1 locus comprise a modification such that theactivity and/or level of the encoded ApoE polypeptide, the interleukin-2receptor gamma polypeptide, the Rag2 polypeptide, the Rag1 polypeptide,or both the Rag1 and Rag2 polypeptide is increased. By “increased” isintended any increase in the level or activity of the gene/polypeptideencoded at the locus of interest. For example, an increase in activitycan comprise either (1) a statistically significant increase in theoverall level or activity of a given protein (i.e., ApoE, interleukin-2receptor gamma, Rag1, Rag2 or Rag1 and Rag2) including, for example, anincreased level or activity of 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 120% or greater when compared to anappropriate control. Methods to assay for an increase in theconcentration and/or the activity of anyone of the ApoE, Rag1, Rag2 andinterleukin-2 receptor gamma proteins are known in the art.

The genetic modification to the rat ApoE locus, the rat interleukin-2receptor gamma locus, the rat Rag2 locus, the rat Rag1 locus and/or ratRag2/Rag1 locus can comprise a deletion of an endogenous rat nucleicacid sequence at the genomic locus, an insertion of an exogenous nucleicacid at the genomic locus, or a combination thereof. The deletion and/orinsertion can occur anywhere within the given locus as discussedelsewhere herein.

Further embodiments provided herein comprise the modification of one ormore of the rat ApoE locus, the rat interleukin-2 receptor gamma locus,the rat Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locusthrough the replacement of a portion of the rat ApoE locus, theinterleukin-2 receptor gamma locus, Rag2 locus, Rag1 locus and/orRag2/Rag1 locus with the corresponding homologous or orthologous portionof an ApoE locus, an interleukin-2 receptor gamma locus, a Rag2 locus, aRag1 locus and/or a Rag2/Rag1 locus from another organism.

Still other embodiments, the modification of one or more of the rat ApoElocus, the rat interleukin-2 receptor gamma locus, Rag2 locus, Rag1locus, and/or Rag2/Rag1 locus is carried out through the replacement ofa portion of the rat ApoE locus, the rat interleukin-2 receptor gammalocus and/or the rat Rag2 locus, and/or the Rag1 locus and/or Rag2/Rag1locus with an insert polynucleotide sharing across its full length least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a portionof an ApoE locus, an interleukin-2 receptor gamma locus, a Rag2 locus, aRag1 locus and/or a Rag2/Rag1 locus it is replacing.

The given insert polynucleotide and/or the corresponding region of therat locus being deleted can be a coding region, an intron, an exon, anuntranslated region, a regulatory region, a promoter, or an enhancer orany combination thereof or any portion thereof. Moreover, the giveninsert polynucleotide and/or the region of the rat locus being deletedcan be of any desired length, including for example, between 10-100nucleotides in length, 100-500 nucleotides in length, 500-1 kbnucleotide in length, 1 Kb to 1.5 kb nucleotide in length, 1.5 kb to 2kb nucleotides in length, 2 kb to 2.5 kb nucleotides in length, 2.5 kbto 3 kb nucleotides in length, 3 kb to 5 kb nucleotides in length, 5 kbto 8 kb nucleotides in length, 8 kb to 10 kb nucleotides in length ormore. In other instances, the size of the insertion or replacement isfrom about 5 kb to about 10 kb, from about 10 kb to about 20 kb, fromabout 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kbto about 250 kb, from about 250 kb to about 300 kb, from about 300 kb toabout 350 kb, from about 350 kb to about 400 kb, from about 400 kb toabout 800 kb, from about 800 kb to 1 Mb, from about 300 kb to about 400kb, from about 400 kb to about 500 kb, from about 500 kb to 1 Mb, fromabout 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about2 Mb to about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb, from about 2.8Mb to about 3 Mb. In other embodiments, the given insert polynucleotideand/or the region of the rat locus being deleted is at least 100, 200,300, 400, 500, 600, 700, 800, or 900 nucleotides or at least 1 kb, 2 kb,3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14kb, 15 kb, 16 kb or greater.

The given insert polynucleotide can be from any organism, including, forexample, a rodent, a rat, a mouse, a hamster, a mammal, a non-humanmammal, a human, an agricultural animal or a domestic animal.

As discussed in further detail herein, various methods are provided togenerate targeted modifications of any rat locus of interest, includingfor example, targeted modifications in the rat ApoE locus, the ratinterleukin-2 receptor gamma locus, the rat Rag2 locus, the rat Rag1locus, and/or the rat Rag2/Rag1 locus. Further provided are geneticallymodified rats or genetically modified pluripotent rat cells (e.g., anrat ES cells), which comprise a deletion, an insertion, a replacementand/or any combination thereof at the interleukin-2 receptor gammalocus, at the ApoE locus, at the rat Rag2 locus, at the rat Rag1 locus,and/or at the rat Rag2/Rag1 locus. Such genetic modifications (includingthose that result in an absence, a decrease, an increase or a modulationin activity of the target locus) and are also capable of beingtransmitted through the germline. In specific embodiments, the geneticmodifications result in a knockout of the desired target locus. Suchrats find use in in a variety of experimental systems as discussedelsewhere herein.

For example, ApoE (Apolipoprotein E) knockouts in rats offer an animalmodel to study endothelial function, including, but not limited to,plaque formation, transcriptional changes (Whole Transcriptome ShotgunSequencing (RNA-Seq), and ex vivo function. Moreover, the larger size ofrats facilitate all these assays and potentially improve the quality ofthe RNA-Seq data. ApoE is an important transport molecule and cantransport lipids, such as cholesterol, through the bloodstream. ApoE canalso function in the nervous system, for example, to clear β-amyloidfrom the brain. Modifications in ApoE have been implicated in variousconditions, including, for example, atherosclerosis, hyperlipidemia, andAlzheimer's disease. ApoE knockout animals display impaired clearing oflipoproteins from the blood and develop atherosclerosis. Thus, ApoEknockout animals provide a model to study conditions and/or processessuch as, for example, endothelia function, plaque formation,transcriptional changes (RNA-Seq), hyperlipidemia, atherosclerosis andAlzheimer's disease. Assays to measure ApoE activity are known in theart. For example, a decrease in ApoE activity can be measured byassaying for a decrease in the ApoE levels in a blood sample obtainedfrom a subject by immunoassays, such as by ELISA or by Immunoblottingtechniques. Moreover, the large size of rats facilitates all theseassays and improves the quality of the data.

RAG1 (Recombination-Activating Gene 1) and RAG2(Recombination-Activating Gene 2) are enzymes that are part of amulti-subunit complex having VDJ recombination activity and play animportant role in the rearrangement and recombination of immunoglobulinand T-cell receptor genes in lymphocytes. RAG1 and RAG2 induce a doublestranded DNA cleavage to facilitate recombination and join of segmentsof the T cell receptor and B cell receptor (i.e. immunoglobulin) genes.Knockout of RAG1 and/or RAG2 causes a loss of B cells and T cells in theanimal resulting in severe immunodeficiency. RAG1 and/or RAG2 knockoutanimals find use, for example, in studies of xenografts (i.e. human cellxenografts in rats), cancer, vaccine development, autoimmune disease,infectious disease and graft versus host disease (GVHD). Various assaysto measure RAG1 and/or RAG2 activity are known in the art and include,for example, measuring recombination efficiency or assaying for thepresence or absence of B cells and/or T cells in a subject. Moreover,the large size of rats facilitates all these assays and potentiallyimproves the quality of the data.

The IL-2 receptor (IL-2R) is expressed on the surface of certain immunecells and binds to the cytokine interleukin-2 (IL-2). The IL-2R is anintegral membrane protein comprising at least three separate subunitchains, including, an alpha chain (IL-2Ra, CD25), a beta chain (IL-2Rb,CD122) and a gamma chain (IL2-Rg, CD132). The IL-2 receptor gamma (alsoreferred to as IL2r-γ or IL2Rg) chain is a common gamma chain that isshared by various cytokine receptors, including, for example, thereceptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-2Rg comprisesan ectodomain on the extracellular surface of the cell, whichcontributes to the binding of the ligand, a transmembrane domain, and anintracellular domain which can interact with various molecules to induceintracellular signal transduction pathways. The Il2rg gene is found onthe X-chromosome in mammals and certain mutations in the gamma chaingene in humans can cause human X-linked severe combined immunodeficiency(XSCID) characterized by a profound T-cell defect. In addition, thegamma chain ecto-domain can be shed off of the transmembrane receptorand released as a soluble gamma chain receptor. The soluble gamma chainreceptor can be detected in the blood of a subject and can function toregulate cytokine signaling.

In some embodiments, the rat IL-2Rg chain is replaced with the humanIL2-Rg chain such that the rat expresses a fully human IL-2Rg chain. Inother instances, it may be useful to replace only the ectodomain of therat IL-2Rg chain with the ectodomain of the human IL-2Rg chain. In suchcases, the resulting humanized IL-2Rg chain expressed in a rat comprisesa human ectodomain, with the remainder of the molecule being from therat.

The full-length humanization of IL-2Rg is useful because rats havingthis modified locus will produce human IL-2Rg. This will allow for thedetection of human IL-2Rg in rats with antibodies specific to humanIL-2Rg. The ecto-humanization (i.e., replacing the rat ecto-domain ofIL-2Rg with the human ecto-domain of IL-2Rg) will result in an IL-2Rgpolypeptide that will bind the human ligands for IL2-Rg, but because thecytoplasmic domain is still rat, it ecto-humanized form of IL-2Rg willalso interact with the rat signaling machinery.

2. Modifying a Rat Target Locus

A. Targeting Vectors and Insert Nucleic Acids

i. Insert Nucleic Acid

As used herein, the “insert nucleic acid” comprises a segment of DNAthat one desires to integrate at the target locus. In one embodiment,the insert nucleic acid comprises one or more polynucleotides ofinterest. In other embodiments, the insert nucleic acid can comprise oneor more expression cassettes. A given expression cassette can comprise apolynucleotide of interest, a polynucleotide encoding a selection markerand/or a reporter gene along with the various regulatory components thatinfluence expression. Non-limiting examples of polynucleotides ofinterest, selection markers, and reporter genes that can be includedwithin the insert nucleic acid are discussed in detail elsewhere herein.

In specific embodiments, the insert nucleic acid can comprise a nucleicacid from rat, which can include a segment of genomic DNA, a cDNA, aregulatory region, or any portion or combination thereof. In otherembodiments, the insert nucleic acid can comprise a nucleic acid from anon-human mammal, a rodent, a human, a rat, a mouse, a hamster a rabbit,a pig, a bovine, a deer, a sheep, a goat, a chicken, a cat, a dog, aferret, a primate (e.g., marmoset, rhesus monkey), domesticated mammalor an agricultural mammal or any other organism of interest. As outlinedin further detail herein, the insert nucleic acid employed in thevarious methods and compositions can result in the “humanization” of thea target locus comprising a rat nucleic acid.

In one embodiment, the insert nucleic acid comprises a knock-in alleleof at least one exon of an endogenous gene. In one embodiment, theinsert nucleic acid comprises a knock-in allele of the entire endogenousgene (i.e., “gene-swap knock-in”).

In one embodiment, the insert nucleic acid comprises a regulatoryelement, including for example, a promoter, an enhancer, or atranscriptional repressor-binding element.

In further embodiments, the insert nucleic acid comprises a conditionalallele. In one embodiment, the conditional allele is a multifunctionalallele, as described in US 2011/0104799, which is incorporated byreference in its entirety. In specific embodiments, the conditionalallele comprises: (a) an actuating sequence in sense orientation withrespect to transcription of a target gene, and a drug selection cassettein sense or antisense orientation; (b) in antisense orientation anucleotide sequence of interest (NSI) and a conditional by inversionmodule (COIN, which utilizes an exon-splitting intron and an invertiblegenetrap-like module; see, for example, US 2011/0104799, which isincorporated by reference in its entirety); and (c) recombinable unitsthat recombine upon exposure to a first recombinase to form aconditional allele that (i) lacks the actuating sequence and the DSC,and (ii) contains the NSI in sense orientation and the COIN in antisenseorientation.

The insert nucleic acid ranges from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, or from about 350 kb toabout 400 kb.

In one embodiment, the insert nucleic acid comprises a deletion of a ratgenomic DNA sequence ranging from about 1 kb to about 200 kb, from about2 kb to about 20 kb, or from about 0.5 kb to about 3 Mb. In oneembodiment, the extent of the deletion of the genomic DNA sequence isgreater than a total length of the 5′ homology arm and the 3′ homologyarm. In one embodiment, the extent of the deletion of the genomic DNAsequence ranges from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 150 kb, from about 150 kb to about 200kb, from about 20 kb to about 30 kb, from about 30 kb to about 40 kb,from about 40 kb to about 50 kb, from about 50 kb to about 60 kb, fromabout 60 kb to about 70 kb, from about 70 kb to about 80 kb, from about80 kb to about 90 kb, from about 90 kb to about 100 kb, from about 100kb to about 110 kb, from about 110 kb to about 120 kb, from about 120 kbto about 130 kb, from about 130 kb to about 140 kb, from about 140 kb toabout 150 kb, from about 150 kb to about 160 kb, from about 160 kb toabout 170 kb, from about 170 kb to about 180 kb, from about 180 kb toabout 190 kb, from about 190 kb to about 200 kb, from about 200 kb toabout 250 kb, from about 250 kb to about 300 kb, from about 300 kb toabout 350 kb, from about 350 kb to about 400 kb, from about 400 kb toabout 800 kb, from about 800 kb to 1 Mb, from about 1 Mb to about 1.5Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb, to about 2.5 Mb,from about 2.5 Mb to about 2.8 Mb, from about 2.8 Mb to about 3 Mb, fromabout 200 kb to about 300 kb, from about 300 kb to about 400 kb, fromabout 400 kb to about 500 kb, from about 500 kb to about 1 Mb, fromabout 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb.

In one embodiment, the insert nucleic acid comprises an insertion or areplacement of a rat nucleic acid sequence with a homologous ororthologous human nucleic acid sequence. In one embodiment, the insertnucleic acid comprises an insertion or replacement of a rat DNA sequencewith a homologous or orthologous human nucleic acid sequence at anendogenous rat locus that comprises the corresponding rat DNA sequence.

In one embodiment, the genetic modification is an addition of a nucleicacid sequence. In one embodiment, the added nucleotide sequence rangesfrom 5 kb to 200 kb.

In one embodiment, the insert nucleic acid comprises a geneticmodification in a coding sequence. In one embodiment, the geneticmodification comprises a deletion mutation of a coding sequence. In oneembodiment, the genetic modification comprises a fusion of twoendogenous coding sequences.

In one embodiment, the insert nucleic acid comprises an insertion or areplacement of a rat nucleic acid sequence with a homologous ororthologous human nucleic acid sequence. In one embodiment, the insertnucleic acid comprises an insertion or replacement of a rat DNA sequencewith a homologous or orthologous human nucleic acid sequence at anendogenous rat locus that comprises the corresponding rat DNA sequence.

In one embodiment, the genetic modification comprises a deletion of anon-protein-coding sequence, but does not comprise a deletion of aprotein-coding sequence. In one embodiment, the deletion of thenon-protein-coding sequence comprises a deletion of a regulatoryelement. In one embodiment, the genetic modification comprises adeletion of a promoter. In one embodiment, the genetic modificationcomprises an addition of a promoter or a regulatory element. In oneembodiment, the genetic modification comprises a replacement of apromoter or a regulatory element.

In one embodiment, the nucleic acid sequence of the targeting vector cancomprise a polynucleotide that when integrated into the genome willproduce a genetic modification of a region of the rat ApoE locus,wherein the genetic modification at the ApoE locus results in a decreasein ApoE activity, increase in ApoE activity, or a modulation of ApoEactivity. In one embodiment, an ApoE knockout (“null allele”) isgenerated.

In one embodiment, the nucleic acid sequence of the targeting vector cancomprise a polynucleotide that when integrated into the genome willproduce a genetic modification of a region of the rat interleukin-2receptor locus, wherein the genetic modification at the interleukin-2receptor locus results in a decrease in interleukin-2 receptor activity.In one embodiment, an interleukin-2 receptor knockout (“null allele”) isgenerated.

In further embodiments, the insert nucleic acid results in thereplacement of a portion of the rat ApoE locus, the interleukin-2receptor gamma locus and/or Rag2 locus, and/or Rag1 locus and/orRag2/Rag1 locus with the corresponding homologous or orthologous portionof an ApoE locus, an interleukin-2 receptor gamma locus, a Rag2 locus, aRag1 locus and/or a Rag2/Rag1 locus from another organism.

Still other embodiments, the insert nucleic acid comprises apolynucleotide sharing across its full length least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a portion of an ApoE locus, aninterleukin-2 receptor gamma locus, a Rag2 locus, a Rag1 locus and/or aRag2/Rag1 locus it is replacing.

The given insert polynucleotide and the corresponding region of the ratlocus being replaced can be a coding region, an intron, an exon, anuntranslated region, a regulatory region, a promoter, or an enhancer orany combination thereof. Moreover, the given insert polynucleotideand/or the region of the rat locus being deleted can be of any desiredlength, including for example, between 10-100 nucleotides in length,100-500 nucleotides in length, 500-1 kb nucleotide in length, 1 Kb to1.5 kb nucleotide in length, 1.5 kb to 2 kb nucleotides in length, 2 kbto 2.5 kb nucleotides in length, 2.5 kb to 3 kb nucleotides in length, 3kb to 5 kb nucleotides in length, 5 kb to 8 kb nucleotides in length, 8kb to 10 kb nucleotides in length or more. In other instances, the sizeof the insertion or replacement is from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, from about 350 kb toabout 400 kb, from about 400 kb to about 800 kb, from about 800 kb to 1Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,from about 2 Mb, to about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb,from about 2.8 Mb to about 3 Mb. In other embodiments, the given insertpolynucleotide and/or the region of the rat locus being deleted is atleast 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides or atleast 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or greater.

In one embodiment, the promoter is constitutively active promoter.

In one embodiment, the promoter is an inducible promoter. In oneembodiment, the inducible promoter is a chemically-regulated promoter.In one embodiment, the chemically-regulated promoter is analcohol-regulated promoter. In one embodiment, the alcohol-regulatedpromoter is an alcohol dehydrogenase (alcA) gene promoter. In oneembodiment, the chemically-regulated promoter is atetracycline-regulated promoter. In one embodiment, thetetracycline-regulated promoter is a tetracycline-responsive promoter.In one embodiment, the tetracycline-regulated promoter is a tetracyclineoperator sequence (tetO). In one embodiment, the tetracycline-regulatedpromoter is a tet-On promoter. In one embodiment, thetetracycline-regulated promoter a tet-Off promoter. In one embodiment,the chemically-regulated promoter is a steroid regulated promoter. Inone embodiment, the steroid regulated promoter is a promoter of a ratglucocorticoid receptor. In one embodiment, the steroid regulatedpromoter is a promoter of an estrogen receptor. In one embodiment, thesteroid-regulated promoter is a promoter of an ecdysone receptor. In oneembodiment, the chemically-regulated promoter is a metal-regulatedpromoter. In one embodiment, the metal-regulated promoter is ametalloprotein promoter. In one embodiment, the inducible promoter is aphysically-regulated promoter. In one embodiment, thephysically-regulated promoter is a temperature-regulated promoter. Inone embodiment, the temperature-regulated promoter is a heat shockpromoter. In one embodiment, the physically-regulated promoter is alight-regulated promoter. In one embodiment, the light-regulatedpromoter is a light-inducible promoter. In one embodiment, thelight-regulated promoter is a light-repressible promoter.

In one embodiment, the promoter is a tissue-specific promoter. In oneembodiment, the promoter is a neuron-specific promoter. In oneembodiment, the promoter is a glia-specific promoter. In one embodiment,the promoter is a muscle cell-specific promoter. In one embodiment, thepromoter is a heart cell-specific promoter. In one embodiment, thepromoter is a kidney cell-specific promoter. In one embodiment, thepromoter is a bone cell-specific promoter. In one embodiment, thepromoter is an endothelial cell-specific promoter. In one embodiment,the promoter is an immune cell-specific promoter. In one embodiment, theimmune cell promoter is a B cell promoter. In one embodiment, the immunecell promoter is a T cell promoter.

In one embodiment, the promoter is a developmentally-regulated promoter.In one embodiment, the developmentally-regulated promoter is active onlyduring an embryonic stage of development. In one embodiment, thedevelopmentally-regulated promoter is active only in an adult cell.

In some embodiments, the insert nucleic acid comprises a nucleic acidflanked with site-specific recombination target sequences. It isrecognized the while the entire insert nucleic acid can be flanked bysuch site-specific recombination target sequences, any region orindividual polynucleotide of interest within the insert nucleic acid canalso be flanked by such sites. The site-specific recombinase can beintroduced into the cell by any means, including by introducing therecombinase polypeptide into the cell or by introducing a polynucleotideencoding the site-specific recombinase into the host cell. Thepolynucleotide encoding the site-specific recombinase can be locatedwithin the insert nucleic acid or within a separate polynucleotide. Thesite-specific recombinase can be operably linked to a promoter active inthe cell including, for example, an inducible promoter, a promoter thatis endogenous to the cell, a promoter that is heterologous to the cell,a cell-specific promoter, a tissue-specific promoter, or a developmentalstage-specific promoter. Site-specific recombination target sequences,which can flank the insert nucleic acid or any polynucleotide ofinterest in the insert nucleic acid can include, but are not limited to,loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71,attp, att, FRT, rox, and a combination thereof.

In some embodiments, the site-specific recombination sites flank apolynucleotide encoding a selection marker and/or a reporter genecontained within the insert nucleic acid. In such instances followingintegration of the insert nucleic acid at the targeted locus thesequences between the site-specific recombination sites can be removed.

In one embodiment, the insert nucleic acid comprises a polynucleotideencoding a selection marker. The selection marker can be contained in aselection cassette. Such selection markers include, but are not limited,to neomycin phosphotransferase (neo^(r)), hygromycin Bphosphotransferase (hyg^(r)), puromycin-N-acetyltransferase (puro^(r)),blasticidin S deaminase (bse), xanthine/guanine phosphoribosyltransferase (gpt), or herpes simplex virus thymidine kinase (HSV-k), ora combination thereof. In one embodiment, the polynucleotide encodingthe selection marker is operably linked to a promoter active in thecell, rat cell, pluripotent rat cell or the ES rat cell. When seriallystacking polynucleotides of interest into a targeted locus, theselection marker can comprise a recognition site for a nuclease agent,as outlined above. In one embodiment, the polynucleotide encoding theselection marker is flanked with a site-specific recombination targetsequences.

The insert nucleic acid can further comprise a reporter gene operablylinked to a promoter, wherein the reporter gene encodes a reporterprotein selected from the group consisting of or comprising LacZ, mPlum,mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine,Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald,enhanced green fluorescent protein (EGFP), CyPet, cyan fluorescentprotein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase,and/or a combination thereof. Such reporter genes can be operably linkedto a promoter active in the cell. Such promoters can be an induciblepromoter, a promoter that is endogenous to the reporter gene or thecell, a promoter that is heterologous to the reporter gene or to thecell, a cell-specific promoter, a tissue-specific promoter, or adevelopmental stage-specific promoter.

In one embodiment, nucleic acid insert can comprise a mammalian nucleicacid comprises a genomic locus that encodes a protein expressed in thenervous system, the skeletal system, the digestive system, thecirculatory system, the muscular system, the respiratory system, thecardiovascular system, the lymphatic system, the endocrine system, theurinary system, the reproductive system, or a combination thereof. Inone embodiment, the mammalian nucleic acid comprises a genomic locusthat encodes a protein expressed in a bone marrow or a bonemarrow-derived cell. In one embodiment, the nucleic acid comprises agenomic locus that encodes a protein expressed in a spleen cell.

In one embodiment, the mammalian nucleic acid comprises a genomic locusthat encodes a protein expressed in the nervous system, the skeletalsystem, the digestive system, the circulatory system, the muscularsystem, the respiratory system, the cardiovascular system, the lymphaticsystem, the endocrine system, the urinary system, the reproductivesystem, or a combination thereof. In one embodiment, the mammaliannucleic acid comprises a genomic locus that encodes a protein expressedin a bone marrow or a bone marrow-derived cell. In one embodiment, thenucleic acid comprises a genomic locus that encodes a protein expressedin a spleen cell. In one embodiment, the genomic locus comprises a mousegenomic DNA sequence, a rat genomic DNA sequence a human genomic DNAsequence, or a combination thereof. In one embodiment, the genomic locuscomprises, in any order, rat and human genomic DNA sequences. In oneembodiment, the genomic locus comprises, in any order, mouse and humangenomic DNA sequences. In one embodiment, the genomic locus comprises,in any order, mouse and rat genomic DNA sequences. In one embodiment,the genomic locus comprises, in any order, rat, mouse, and human genomicDNA sequences.

In one embodiment, the genomic locus comprises a mouse genomic DNAsequence, a rat genomic DNA sequence a human genomic DNA sequence, or acombination thereof. In one embodiment, the genomic locus comprises, inany order, rat and human genomic DNA sequences. In one embodiment, thegenomic locus comprises, in any order, mouse and human genomic DNAsequences. In one embodiment, the genomic locus comprises, in any order,mouse and rat genomic DNA sequences. In one embodiment, the genomiclocus comprises, in any order, rat, mouse, and human genomic DNAsequences.

In one embodiment, the genetic modification comprises at least one humandisease allele of a human gene. In one embodiment, the human disease isa neurological disease. In one embodiment, the human disease is acardiovascular disease. In one embodiment, the human disease is a kidneydisease. In one embodiment, the human disease is a muscle disease. Inone embodiment, the human disease is a blood disease. In one embodiment,the human disease is a cancer. In one embodiment, the human disease isan immune system disease.

In one embodiment, the human disease allele is a dominant allele. In oneembodiment, the human disease allele is a recessive allele. In oneembodiment, the human disease allele comprises a single nucleotidepolymorphism (SNP) allele.

In one embodiment, the genetic modification produces a mutant form of aprotein with an altered binding characteristic, altered localization,altered expression, and/or altered expression pattern.

In one embodiment, the insert nucleic acid comprises a selectioncassette. In one embodiment, the selection cassette comprises a nucleicacid sequence encoding a selective marker, wherein the nucleic acidsequence is operably linked to a promoter active in rat ES cells. In oneembodiment, the selective marker is selected from or comprises ahygromycin resistance gene or a neomycin resistance gene.

In one embodiment, the nucleic acid comprises a genomic locus thatencodes a protein expressed in a B cell. In one embodiment, the nucleicacid comprises a genomic locus that encodes a protein expressed in animmature B cell. In one embodiment, the nucleic acid comprises a genomiclocus that encodes a protein expressed in a mature B cell.

In one embodiment, the insert nucleic acid comprises a regulatoryelement. In one embodiment, the regulatory element is a promoter. In oneembodiment, the regulatory element is an enhancer. In one embodiment,the regulatory element is a transcriptional repressor-binding element.

In one embodiment, the genetic modification comprises a deletion of anon-protein-coding sequence, but does not comprise a deletion of aprotein-coding sequence. In one embodiment, the deletion of thenon-protein-coding sequence comprises a deletion of a regulatoryelement. In one embodiment, the genetic modification comprises adeletion of a regulatory element. In one embodiment, the geneticmodification comprises an addition of a promoter or a regulatoryelement. In one embodiment, the genetic modification comprises areplacement of a promoter or a regulatory element.

ii. Expression Cassettes

Provided herein are polynucleotides or nucleic acid molecules comprisingthe various components employed in a targeted genomic integration systemprovided herein (i.e. any one of or any combination of nuclease agents,recognition sites, insert nucleic acids, polynucleotides of interest,targeting vectors, selection markers, and other components).

The terms “polynucleotide,” “polynucleotide sequence,” “nucleic acidsequence,” and “nucleic acid fragment” are used interchangeably herein.These terms encompass nucleotide sequences and the like. Apolynucleotide may be a polymer of RNA or DNA that is single- ordouble-stranded, that optionally contains synthetic, non-natural oraltered nucleotide bases. A polynucleotide in the form of a polymer ofDNA may be comprised of one or more segments of cDNA, genomic DNA,synthetic DNA, or mixtures thereof. Polynucleotides can comprisedeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues, and any combination these.The polynucleotides provided herein also encompass all forms ofsequences including, but not limited to, single-stranded forms,double-stranded forms, hairpins, stem-and-loop structures, and the like.

Further provided are recombinant polynucleotides comprising the variouscomponents of the targeted genomic integration system. The terms“recombinant polynucleotide” and “recombinant DNA construct” are usedinterchangeably herein. A recombinant construct comprises an artificialor heterologous combination of nucleic acid sequences, e.g., regulatoryand coding sequences that are not found together in nature. In otherembodiments, a recombinant construct may comprise regulatory sequencesand coding sequences that are derived from different sources, orregulatory sequences and coding sequences derived from the same source,but arranged in a manner different than that found in nature. Such aconstruct may be used by itself or may be used in conjunction with avector. If a vector is used, then the choice of vector is dependent uponthe method that is used to transform the host cells as is well known tothose skilled in the art. For example, a plasmid vector can be used.Genetic elements required to successfully transform, select, andpropagate host cells comprising any of the isolated nucleic acidfragments provided herein are also provided. Screening may beaccomplished by Southern analysis of DNA, Northern analysis of mRNAexpression, immunoblotting analysis of protein expression, or phenotypicanalysis, among others.

In specific embodiments, one or more of the components of the targetedgenomic integration system described herein can be provided in anexpression cassette for expression in a prokaryotic cell, a eukaryoticcell, a bacterial, a yeast cell, or a mammalian cell or other organismor cell type of interest. The cassette can include 5′ and 3′ regulatorysequences operably linked to a polynucleotide provided herein. “Operablylinked” comprises a relationship wherein the components operably linkedfunction in their intended manner. For example, an operable linkagebetween a polynucleotide of interest and a regulatory sequence (i.e., apromoter) is a functional link that allows for expression of thepolynucleotide of interest. Operably linked elements may be contiguousor non-contiguous. When used to refer to the joining of two proteincoding regions, operably linked means that the coding regions are in thesame reading frame. In another instance, a nucleic acid sequenceencoding a protein may be operably linked to regulatory sequences (e.g.,promoter, enhancer, silencer sequence, etc.) so as to retain propertranscriptional regulation. In one instance, a nucleic acid sequence ofan immunoglobulin variable region (or V(D)J segments) may be operablylinked to a nucleic acid sequence of an immunoglobulin constant regionso as to allow proper recombination between the sequences into animmunoglobulin heavy or light chain sequence.

The cassette may additionally contain at least one additionalpolynucleotide of interest to be co-introduced into the organism.Alternatively, the additional polynucleotide of interest can be providedon multiple expression cassettes. Such an expression cassette isprovided with a plurality of restriction sites and/or recombinationsites for insertion of a recombinant polynucleotide to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selection marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a recombinant polynucleotide provided herein, and atranscriptional and translational termination region (i.e., terminationregion) functional in mammalian cell or a host cell of interest. Theregulatory regions (i.e., promoters, transcriptional regulatory regions,and translational termination regions) and/or a polynucleotide providedherein may be native/analogous to the host cell or to each other.Alternatively, the regulatory regions and/or a polynucleotide providedherein may be heterologous to the host cell or to each other. Forexample, a promoter operably linked to a heterologous polynucleotide isfrom a species different from the species from which the polynucleotidewas derived, or, if from the same/analogous species, one or both aresubstantially modified from their original form and/or genomic locus, orthe promoter is not the native promoter for the operably linkedpolynucleotide. Alternatively, the regulatory regions and/or arecombinant polynucleotide provided herein may be entirely synthetic.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked recombinantpolynucleotide, may be native with the host cell, or may be derived fromanother source (i.e., foreign or heterologous) to the promoter, therecombinant polynucleotide, the host cell, or any combination thereof.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation. Toward this end, adapters or linkers may be employed tojoin the DNA fragments or other manipulations may be involved to providefor convenient restriction sites, removal of superfluous DNA, removal ofrestriction sites, or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g.,transitions and transversions, may be involved.

A number of promoters can be used in the expression cassettes providedherein. The promoters can be selected based on the desired outcome. Itis recognized that different applications can be enhanced by the use ofdifferent promoters in the expression cassettes to modulate the timing,location and/or level of expression of the polynucleotide of interest.Such expression constructs may also contain, if desired, a promoterregulatory region (e.g., one conferring inducible, constitutive,environmentally- or developmentally-regulated, or cell- ortissue-specific/selective expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, a transcriptiontermination site, and/or a polyadenylation signal.

The expression cassette containing the polynucleotides provided hereincan also comprise a selection marker gene for the selection oftransformed cells. Selectable marker genes are utilized for theselection of transformed cells or tissues.

Where appropriate, the sequences employed in the methods andcompositions (i.e., the polynucleotide of interest, the nuclease agent,etc.) may be optimized for increased expression in the cell. That is,the genes can be synthesized using codons preferred in a given cell ofinterest including, for example, mammalian-preferred codons,human-preferred codons, rodent-preferred codon, mouse-preferred codons,rat-preferred codons, etc. for improved expression.

The various methods and compositions provided herein can employselection markers. Various selection markers can be used in the methodsand compositions disclosed herein. Such selection markers can, forexample, impart resistance to an antibiotic such as G418, hygromycin,blasticidin, neomycin, or puromycin. Such selection markers includeneomycin phosphotransferase (neo^(r)), hygromycin B phosphotransferase(hyg^(r)), puromycin-N-acetyltransferase (puro^(r)), and blasticidin Sdeaminase (bsr^(r)). In still other embodiments, the selection marker isoperably linked to an inducible promoter and the expression of theselection marker is toxic to the cell. Non-limiting examples of suchselection markers include xanthine/guanine phosphoribosyl transferase(gpt), hahypoxanthine-guanine phosphoribosyltransferase (HGPRT) orherpes simplex virus thymidine kinase (HSV-TK). The polynucleotideencoding the selection markers are operably linked to a promoter activein the cell.

iii. Targeting Vectors

Targeting vectors are employed to introduce the insert nucleic acid intothe target locus of the rat nucleic acid. The targeting vector comprisesthe insert nucleic acid and further comprises a 5′ and a 3′ homologyarm, which flank the insert nucleic acid. The homology arms, which flankthe insert nucleic acid, correspond to regions within the target locusof the rat nucleic acid. For ease of reference, the correspondingcognate genomic regions within the targeted genomic locus are referredto herein as “target sites”. For example, a targeting vector cancomprise a first insert nucleic acid flanked by a first and a secondhomology arm complementary to a first and a second target site. As such,the targeting vector thereby aids in the integration of the insertnucleic acid into the target locus of the rat nucleic acid through ahomologous recombination event that occurs between the homology arms andthe complementary target sites within the genome of the cell.

In one embodiment, the target locus of the rat nucleic acid comprises afirst nucleic acid sequence that is complementary to the 5′ homology armand a second nucleic acid sequence that is complementary to the 3′homology arm. In one embodiment, the first and the second nucleic acidsequences are separated by at least 5 kb. In another embodiment, thefirst and the second nucleic acid sequences are separated by at least 5kb but less than 200 kb. In one embodiment, the first and the secondnucleic acid sequences are separated by at least 10 kb. In oneembodiment, the first and the second nucleic acid sequences areseparated by at least 20 kb, at least 30 kb, at least 40 kb, at least 50kb, at least 60 kb, at least 70 kb, at least 80 kb, at least 90 kb, atleast 100 kb, at least 110 kb, at least 120 kb, at least 130 kb, atleast 140 kb, at least 150 kb, at least 160 kb, at least 170 kb, atleast 180 kb, at least 190 kb, or at least 200 kb. In still furtherembodiments, the first and the second nucleic acid sequence is separatedby at least 5 kb but less than 10 kb, at least 5 kb but less than 3 Mb,at least 10 kb but less than 20 kb, at least 20 kb but less than 40 kb,at least 40 kb but less than 60 kb, at least 60 kb but less than 80 kb,at least about 80 kb but less than 100 kb, at least 100 kb but less than150 kb, or at least 150 kb but less than 200 kb, at least about 200 kbbut less than about 300 kb, at least about 300 kb but less than about400 kb, at least about 400 kb but less than about 500 kb, at least about500 kb but less than about 1 Mb, at least about 1.5 Mb but less thanabout 2 Mb, at least about 1 Mb but less than about 1.5 Mb, at leastabout 2 Mb but less than 2.5 Mb, at least about 2.5 Mb but less than 3Mb, or at least about 2 Mb but less than about 3 Mb.

A homology arm of the targeting vector can be of any length that issufficient to promote a homologous recombination event with acorresponding target site, including for example, at least 5-10 kb, 5-15kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50 kb, 50-60 kb, 60-70 kb, 70-80kb, 80-90 kb, 90-100 kb, 100-110 kb, 110-120 kb, 120-130 kb, 130-140 kb,140-150 kb, 150-160 kb, 160-170 kb, 170-180 kb, 180-190 kb, 190-200 kbin length or greater. As outlined in further detail below, largetargeting vectors can employ targeting arms of greater length. In aspecific embodiment, the sum total of the 5′ homology arm and the 3′homology arm is at least 10 kb or the sum total of the 5′ homology armand the 3′ homology arm is at least about 16 kb to about 100 kb or about30 kb to about 100 kb. In other embodiments, the size of the sum totalof the total of the 5′ and 3′ homology arms of the LTVEC is about 10 kbto about 150 kb, about 10 kb to about 100 kb, about 10 kb to about 75kb, about 20 kb to about 150 kb, about 20 kb to about 100 kb, about 20kb to about 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100kb, about 30 kb to about 75 kb, about 40 kb to about 150 kb, about 40 kbto about 100 kb, about 40 kb to about 75 kb, about 50 kb to about 150kb, about 50 kb to about 100 kb, or about 50 kb to about 75 kb, about 10kb to about 30 kb, about 20 kb to about 40 kb, about 40 kb to about 60kb, about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100kb to about 120 kb, or from about 120 kb to about 150 kb. In oneembodiment, the size of the deletion is the same or similar to the sizeof the sum total of the 5′ and 3′ homology arms of the LTVEC.

When nuclease agents are employed, the cognate genomic regionscorresponding to the 5′ and 3′ homology arms of a targeting vector are“located in sufficient proximity” to nuclease target sites so as topromote the occurrence of a homologous recombination event between thecognate genomic regions and the homology arms upon a nick ordouble-strand break at the recognition site. For example, the nucleasetarget sites can be located anywhere between the cognate genomic regionscorresponding to the 5′ and 3′ homology arms. In specific embodiments,the recognition site is immediately adjacent to at least one or both ofthe cognate genomic regions.

As used herein, a homology arm and a target site (i.e., cognate genomicregion) “complement” or are “complementary” to one another when the tworegions share a sufficient level of sequence identity to one another toact as substrates for a homologous recombination reaction. By “homology”is meant DNA sequences that are either identical or share sequenceidentity to a corresponding or “complementary” sequence. The sequenceidentity between a given target site and the corresponding homology armfound on the targeting vector can be any degree of sequence identitythat allows for homologous recombination to occur. For example, theamount of sequence identity shared by the homology arm of the targetingvector (or a fragment thereof) and the target site (or a fragmentthereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% sequence identity, such that the sequences undergohomologous recombination. Moreover, a complementary region of homologybetween the homology arm and the complementary target site can be of anylength that is sufficient to promote homologous recombination at thecleaved recognition site. For example, a given homology arm and/orcomplementary target site can comprise complementary regions of homologythat are at least 5-10 kb, 5-15 kb, 10-20 kb, 20-30 kb, 30-40 kb, 40-50kb, 50-60 kb, 60-70 kb, 70-80 kb, 80-90 kb, 90-100 kb, 100-110 kb,110-120 kb, 120-130 kb, 130-140 kb, 140-150 kb, 150-160 kb, 160-170 kb,170-180 kb, 180-190 kb, 190-200 kb in length or greater (such asdescribed in the LTVEC vectors described elsewhere herein) such that thehomology arm has sufficient homology to undergo homologous recombinationwith the corresponding target sites within the genome of the cell. Forease of reference the homology arms are referred to herein as a 5′ and a3′ homology arm. This terminology relates to the relative position ofthe homology arms to the insert nucleic acid within the targetingvector.

The homology arms of the targeting vector are therefore designed to becomplementary to a target site with the targeted locus. Thus, thehomology arms can be complementary to a locus that is native to thecell, or alternatively they can be complementary to a region of aheterologous or exogenous segment of DNA that was integrated into thegenome of the cell, including, but not limited to, transgenes,expression cassettes, or heterologous or exogenous regions of genomicDNA. Alternatively, the homology arms of the targeting vector can becomplementary to a region of a human artificial chromosome or any otherengineered genomic region contained in an appropriate host cell. Stillfurther, the homology arms of the targeting vector can be complementaryto or be derived from a region of a BAC library, a cosmid library, or aP1 phage library. Thus, in specific embodiments, the homology arms ofthe targeting vector are complementary to a rat genomic locus that isnative, heterologous or exogenous to a given cell. In furtherembodiments, the homology arms are complementary to a rat genomic locusthat is not targetable using a conventional method or can be targetedonly incorrectly or only with significantly low efficiency, in theabsence of a nick or double-strand break induced by a nuclease agent. Inone embodiment, the homology arms are derived from a synthetic DNA.

In still other embodiments, the 5′ and 3′ homology arms arecomplementary to the same genome as the targeted genome. In oneembodiment, the homology arms are from a related genome, e.g., thetargeted genome is a rat genome of a first strain, and the targetingarms are from a rat genome of a second strain, wherein the first strainand the second strain are different. In other embodiments, the homologyarms are from the genome of the same animal or are from the genome ofthe same strain, e.g., the targeted genome is a rat genome of a firststrain, and the targeting arms are from a rat genome from the same rator from the same strain.

The targeting vector (such as a large targeting vector) can alsocomprise a selection cassette or a reporter gene as discussed elsewhereherein. The selection cassette can comprise a nucleic acid sequenceencoding a selection marker, wherein the nucleic acid sequence isoperably linked to a promoter. The promoter can be active in aprokaryotic cell of interest and/or active in a eukaryotic cell ofinterest. Such promoters can be an inducible promoter, a promoter thatis endogenous to the reporter gene or the cell, a promoter that isheterologous to the reporter gene or to the cell, a cell-specificpromoter, a tissue-specific promoter or a developmental stage-specificpromoter. In one embodiment, the selection marker is selected from orcomprises neomycin phosphotransferase (neo^(r)), hygromycin Bphosphotransferase (hyg^(r)), puromycin-N-acetyltransferase (puro^(r)),blasticidin S deaminase (bsr^(r)), xanthine/guanine phosphoribosyltransferase (gpt), and herpes simplex virus thymidine kinase (HSV-k),and/or a combination thereof. The selection marker of the targetingvector can be flanked by the 5′ and 3′ homology arms or found either 5′or 3′ to the homology arms.

In one embodiment, the targeting vector (such as a large targetingvector) comprises a reporter gene operably linked to a promoter, whereinthe reporter gene encodes a reporter protein selected from the groupconsisting of or comprises LacZ, mPlum, mCherry, tdTomato, mStrawberry,J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellowfluorescent protein (EYFP), Emerald, enhanced green fluorescent protein(EGFP), CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire,luciferase, alkaline phosphatase, and/or a combination thereof. Suchreporter genes can be operably linked to a promoter active in the cell.Such promoters can be an inducible promoter, a promoter that isendogenous to the report gene or the cell, a promoter that isheterologous to the reporter gene or to the cell, a cell-specificpromoter, a tissue-specific promoter or a developmental stage-specificpromoter.

In one embodiment, combined use of the targeting vector (including, forexample, a large targeting vector) with the nuclease agent results in anincreased targeting efficiency compared to use of the targeting vectoralone. In one embodiment, when the targeting vector is used inconjunction with the nuclease agent, targeting efficiency of thetargeting vector is increased at least by two-fold, at least three-fold,or at least 4-fold when compared to when the targeting vector is usedalone.

When employing a targeting vector, the vector design can be such as toallow for the insertion of a given sequence that is from about 5 kb toabout 200 kb as described herein. In one embodiment, the insertion isfrom about 5 kb to about 10 kb, from about 10 kb to about 20 kb, fromabout 20 kb to about 30 kb, from about 30 kb to about 40 kb, from about40 kb to about 50 kb, from about 50 kb to about 60 kb, from about 60 kbto about 70 kb, from about 80 kb to about 90 kb, from about 90 kb toabout 100 kb, from about 100 kb to about 110 kb, from about 110 kb toabout 120 kb, from about 120 kb to about 130 kb, from about 130 kb toabout 140 kb, from about 140 kb to about 150 kb, from about 150 kb toabout 160 kb, from about 160 kb to about 170 kb, from about 170 kb toabout 180 kb, from about 180 kb to about 190 kb, or from about 190 kb toabout 200 kb, from about 5 kb to about 10 kb, from about 10 kb to about20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb,from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, fromabout 100 kb to about 150 kb, from about 150 kb to about 200 kb, fromabout 200 kb to about 250 kb, from about 250 kb to about 300 kb, fromabout 300 kb to about 350 kb, or from about 350 kb to about 400 kb.

When employing a targeting vector, the vector design can be such as toallow for the replacement of a given sequence that is from about 5 kb toabout 200 kb or from about 5 kb to about 3.0 Mb as described herein. Inone embodiment, the replacement is from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 30 kb, from about30 kb to about 40 kb, from about 40 kb to about 50 kb, from about 50 kbto about 60 kb, from about 60 kb to about 70 kb, from about 80 kb toabout 90 kb, from about 90 kb to about 100 kb, from about 100 kb toabout 110 kb, from about 110 kb to about 120 kb, from about 120 kb toabout 130 kb, from about 130 kb to about 140 kb, from about 140 kb toabout 150 kb, from about 150 kb to about 160 kb, from about 160 kb toabout 170 kb, from about 170 kb to about 180 kb, from about 180 kb toabout 190 kb, from about 190 kb to about 200 kb, from about 5 kb toabout 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, orfrom about 150 kb to about 200 kb, from about 200 kb to about 300 kb,from about 300 kb to about 400 kb, from about 400 kb to about 500 kb,from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, fromabout 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or fromabout 2.5 Mb to about 3 Mb.

In one embodiment, the targeting vector comprises a site-specificrecombinase gene. In one embodiment, the site-specific recombinase geneencodes a Cre recombinase. In one embodiment, the Cre recombinase geneis Crei, wherein two exons encoding the Cre recombinase are separated byan intron to prevent its expression in a prokaryotic cell.

In one embodiment, the Cre recombinase gene further comprises a nuclearlocalization signal to facilitate localization of Cre (or anyrecombinase or nuclease agent) to the nucleus (e.g., the gene is anNL-Cre gene). In a specific embodiment, the Cre recombinase gene furthercomprises a nuclear localization signal and an intron (e.g., NL-Crei).

in various embodiments, a suitable promoter for expression of thenuclease agent (including the Cre or Crei recombinase discussed above)is selected from or comprises a Prm1, Blimp1, Gata6, Gata4, Igf2, Lhx2,Lhx5, and/or Pax3. In a specific embodiment, the promoter is the Gata6or Gata4 promoter. The various promoters can be from any organism,including for example, a rodent such as a mouse or a rat. In anotherspecific embodiment, the promoter is a Prm1 promoter. In anotherspecific embodiment, the promoter is a rat Prm1 promoter. In anotherspecific embodiment, the promoter is a mouse Prm1 promoter. In anotherspecific embodiment, the promoter is a Blimp1 promoter or a fragmentthereof, e.g., a 1 kb or 2 kb fragment of a Blimp1 promoter. See, forexample, U.S. Pat. No. 8,697,851 and U.S. Application Publication2013-0312129, both of which are herein incorporated by reference intheir entirety.

iv. Large Targeting Vectors

The term “large targeting vector” or “LTVEC” as used herein compriseslarge targeting vectors that comprise homology arms that correspond toand are derived from nucleic acid sequences larger than those typicallyused by other approaches intended to perform homologous targeting incells and/or comprising insert nucleic acids comprising nucleic acidsequences larger than those typically used by other approaches intendedto perform homologous recombination targeting in cells. For example, theLTVEC make possible the modification of large loci that cannot beaccommodated by traditional plasmid-based targeting vectors because oftheir size limitations. In specific embodiments, the homology armsand/or the insert nucleic acid of the LTVEC comprises genomic sequenceof a eukaryotic cell. The size of the LTVEC is too large to enablescreening of targeting events by conventional assays, e.g., southernblotting and long-range (e.g., 1 kb-5 kb) PCR. Examples of the LTVEC,include, but are not limited to, vectors derived from a bacterialartificial chromosome (BAC), a human artificial chromosome or a yeastartificial chromosome (YAC). Non-limiting examples of LTVECs and methodsfor making them are described, e.g., in U.S. Pat. Nos. 6,586,251,6,596,541, 7,105,348, and WO 2002/036789 (PCT/US01/45375), and US2013/0137101, each of which is herein incorporated by reference.

The LTVEC can be of any length, including, but not limited to, fromabout 20 kb to about 400 kb, from about 20 kb to about 30 kb, from about30 kb to 40 kb, from about 40 kb to about 50 kb, from about 50 kb toabout 75 kb, from about 75 kb to about 100 kb, from about 100 kb to 125kb, from about 125 kb to about 150 kb, from about 150 kb to about 175kb, about 175 kb to about 200 kb, from about 200 kb to about 225 kb,from about 225 kb to about 250 kb, from about 250 kb to about 275 kb orfrom about 275 kb to about 300 kb, from about 200 kb to about 300 kb,from about 300 kb to about 350 kb, from about 350 kb to about 400 kb,from about 350 kb to about 550 kb. In one embodiment, the LTVEC is about100 kb.

In one embodiment, the LTVEC comprises an insert nucleic acid rangingfrom about 5 kb to about 200 kb, from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 30 kb, from about0.5 kb to about 30 kb, from about 0.5 kb to about 40 kb, from about 30kb to about 150 kb, from about 0.5 kb to about 150 kb, from about 30 kbto about 40 kb, from about 40 kb to about 50 kb, from about 60 kb toabout 70 kb, from about 80 kb to about 90 kb, from about 90 kb to about100 kb, from about 100 kb to about 110 kb, from about 120 kb to about130 kb, from about 130 kb to about 140 kb, from about 140 kb to about150 kb, from about 150 kb to about 160 kb, from about 160 kb to about170 kb, from about 170 kb to about 180 kb, from about 180 kb to about190 kb, or from about 190 kb to about 200 kb, from about 5 kb to about10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, fromabout 80 kb to about 100 kb, from about 100 kb to about 150 kb, fromabout 150 kb to about 200 kb, from about 200 kb to about 250 kb, fromabout 250 kb to about 300 kb, from about 300 kb to about 350 kb, or fromabout 350 kb to about 400 kb;

When employing a LTVEC, the vector design can be such as to allow forthe replacement of a given sequence that is from about 5 kb to about 200kb or from about 5 kb to about 3 Mb as described herein. In oneembodiment, the replacement is from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 30 kb, from about30 kb to about 40 kb, from about 40 kb to about 50 kb, from about 50 kbto about 60 kb, from about 60 kb to about 70 kb, from about 80 kb toabout 90 kb, from about 90 kb to about 100 kb, from about 100 kb toabout 110 kb, from about 110 kb to about 120 kb, from about 120 kb toabout 130 kb, from about 130 kb to about 140 kb, from about 140 kb toabout 150 kb, from about 150 kb to about 160 kb, from about 160 kb toabout 170 kb, from about 170 kb to about 180 kb, from about 180 kb toabout 190 kb, from about 190 kb to about 200 kb, from about 5 kb toabout 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb,from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, orfrom about 150 kb to about 200 kb, from about 200 kb to about 300 kb,from about 300 kb to about 400 kb, from about 400 kb to about 500 kb,from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, fromabout 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or fromabout 2.5 Mb to about 3 Mb.

In one embodiment, the homology arms of the LTVEC are derived from a BAClibrary, a cosmid library, or a P1 phage library. In other embodiments,the homology arms are derived from the targeted genomic locus of thecell and in some instances the target genomic locus, which the LTVEC isdesigned to target is not targetable using a conventional method. Instill other embodiments, the homology arms are derived from a syntheticDNA.

In one embodiment, a sum total of the 5′ homology arm and the 3′homology arm in the LTVEC is at least 10 kb. In other embodiments, thesum total of the 5′ and the 3′ homology arms of the LTVEC is from about10 kb to about 30 kb, from about 20 kb to about 40 kb, from about 40 kbto about 60 kb, from about 60 kb to about 80 kb, from about 80 kb toabout 100 kb, from 100 kb to about 120 kb, from about 120 kb to about140 kb, from about 140 kb to about 160 kb, from about 160 kb to about180 kb, from about 180 kb to about 200 kb. In one embodiment the sumtotal of the 5′ and the 3′ homology arms of the LTVEC is from about 30kb to about 100 kb. In other embodiments, the size of the sum total ofthe total of the 5′ and 3′ homology arms of the LTVEC is about 10 kb toabout 150 kb, about 10 kb to about 100 kb, about 10 kb to about 75 kb,about 20 kb to about 150 kb, about 20 kb to about 100 kb, about 20 kb toabout 75 kb, about 30 kb to about 150 kb, about 30 kb to about 100 kb,about 30 kb to about 75 kb, about 40 kb to about 150 kb, about 40 kb toabout 100 kb, about 40 kb to about 75 kb, about 50 kb to about 150 kb,about 50 kb to about 100 kb, or about 50 kb to about 75 kb, about 10 kbto about 30 kb, about 20 kb to about 40 kb, about 40 kb to about 60 kb,about 60 kb to about 80 kb, about 80 kb to about 100 kb, about 100 kb toabout 120 kb, or from about 120 kb to about 150 kb. In one embodiment,the size of the deletion is the same or similar to the size of the sumtotal of the 5′ and 3′ homology arms of the LTVEC.

In other embodiments, the 5′ homology arm ranges from about 5 kb toabout 100 kb. In one embodiment, the 3′ homology arm ranges from about 5kb to about 100 kb. In other embodiments, the sum total of the 5′ and 3′homology arms are from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 30 kb, from about 30 kb to about40 kb, from about 40 kb to about 50 kb, from about 50 kb to about 60 kb,from about 60 kb to about 70 kb, from about 70 kb to about 80 kb, fromabout 80 kb to about 90 kb, from about 90 kb to about 100 kb, from about100 kb to about 110 kb, from about 110 kb to about 120 kb, from about120 kb to about 130 kb, from about 130 kb to about 140 kb, from about140 kb to about 150 kb, from about 150 kb to about 160 kb, from about160 kb to about 170 kb, from about 170 kb to about 180 kb, from about180 kb to about 190 kb, from about 190 kb to about 200 kb, or from about30 kb to about 100 kb, about 10 kb to about 30 kb, about 20 kb to about40 kb, about 40 kb to about 60 kb, about 60 kb to about 80 kb, about 80kb to about 100 kb, about 100 kb to about 120 kb, or from about 120 kbto about 150 kb.

In one embodiment, the LTVEC comprises an insert nucleic acid that ishomologous or orthologous to a rat nucleic acid sequence flanked by theLTVEC homology arms. In one embodiment, the insert nucleic acid sequenceis from a species other than a rat. In one embodiment, the insertnucleic acid that is homologous or orthologous to the rat nucleic acidsequence is a mammalian nucleic acid. In one embodiment, the mammaliannucleic acid is a mouse nucleic acid. In one embodiment, the mammaliannucleic acid is a human nucleic acid. In one embodiment, the insertnucleic acid is a genomic DNA. In one embodiment, the insert is from 5kb to 200 kb as described above.

In one embodiment, the LTVEC comprises a selection cassette or areporter gene. Various forms of the selection cassette and reporter genethat can be employed are discussed elsewhere herein.

As described elsewhere herein, the LTVEC can also be used in the methodsprovided herein in combination with a nuclease agent that promotes ahomologous recombination between the targeting vector and the targetlocus of a rat nucleic acid in a pluripotent rat cell.

In one embodiment, the large targeting vector (LTVEC) comprises asite-specific recombinase gene. In one embodiment, the site-specificrecombinase gene encodes a Cue recombinase. In one embodiment, the Crerecombinase gene is Crei, wherein two exons encoding the Cre recombinaseare separated by an intron to prevent its expression in a prokaryoticcell. In one embodiment, the Cre recombinase gene further comprises anuclear localization signal to facilitate localization of Cre (or anyrecombinase or nuclease agent) to the nucleus (e.g., the gene is anNL-Cre gene). In a specific embodiment, the Cre recombinase gene furthercomprises a nuclear localization signal and an intron (e.g., NL-Crei)

In various embodiments, a suitable promoter for expression of thenuclease agent (including the Cre or Crei recombinase discussed above)is selected from or comprises a Prm1, Blimp1, Gata6, Gata4, Igf2, Lhx2,Lhx5, and/or Pax3. In a specific embodiment, the promoter is the Gata6or Gata4 promoter. The various promoters can be from any organism,including for example, a rodent such as a mouse or a rat. In anotherspecific embodiment, the promoter is a Prm1 promoter. In anotherspecific embodiment, the promoter is a rat Prm1 promoter. In anotherspecific embodiment, the promoter is a mouse Prm1 promoter. In anotherspecific embodiment, the promoter is a Blimp1 promoter or a fragmentthereof, e.g., a 1 kb or 2 kb fragment of a Blimp1 promoter. See, forexample, U.S. Pat. No. 8,697,851 and U.S. Application Publication2013-0312129, both of which are herein incorporated by reference intheir entirety.

In one embodiment, the LTVEC comprises an insert nucleic acid that canproduce a deletion, addition, replacement or a combination thereof of aregion of the rat ApoE locus, the IL-2Rg locus, the Rag2 locus, the Rag1locus and/or the Rag2/Rag1 locus as discussed in detail elsewhereherein. In specific embodiments, the genetic modification at the ApoElocus results in a decrease, an increase or a modulation in ApoEactivity, IL-2Rg activity, Rag2 activity, Rag1 activity and/or Rag2 andRag1 activity. In one embodiment, an ApoE knockout, and IL-2Rg knockout,a Rag2 knockout, a Rag1 knockout, a Rag2/Rag1 knockout is generated. Asdiscussed below, nuclease agents can be employed with any of the LTVECtargeting systems to target any genomic locus of interest.

v. Nuclease Agents and Recognition Sites for Nuclease Agents

As outlined in detail above, nuclease agents may be utilized in themethods and compositions disclosed herein to aid in the modification ofthe target locus both in a prokaryotic cell or within a pluripotent ratcell. Such a nuclease agent may promote homologous recombination betweenthe targeting vector and the target locus. In one embodiment, thenuclease agent comprises an endonuclease agent.

As used herein, the term “recognition site for a nuclease agent”comprises a DNA sequence at which a nick or double-strand break isinduced by a nuclease agent. The recognition site for a nuclease agentcan be endogenous (or native) to the cell or the recognition site can beexogenous to the cell. In specific embodiments, the recognition site isexogenous to the cell and thereby is not naturally occurring in thegenome of the cell. In still further embodiments, the recognition siteis exogenous to the cell and to the polynucleotides of interest that onedesired to be positioned at the target genomic locus. In furtherembodiments, the exogenous or endogenous recognition site is presentonly once in the genome of the host cell. In specific embodiments, anendogenous or native site that occurs only once within the genome isidentified. Such a site can then be used to design nuclease agents thatwill produce a nick or double-strand break at the endogenous recognitionsite.

The length of the recognition site can vary, and includes, for example,recognition sites that are at least 4, 6, 8, 10, 12, 14, 16, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or morenucleotides in length. In one embodiment, each monomer of the nucleaseagent recognizes a recognition site of at least 9 nucleotides. In otherembodiments, the recognition site is from about 9 to about 12nucleotides in length, from about 12 to about 15 nucleotides in length,from about 15 to about 18 nucleotides in length, or from about 18 toabout 21 nucleotides in length, and any combination of such subranges(e.g., 9-18 nucleotides). The recognition site could be palindromic,that is, the sequence on one strand reads the same in the oppositedirection on the complementary strand. It is recognized that a givennuclease agent can bind the recognition site and cleave that bindingsite or alternatively, the nuclease agent can bind to a sequence that isthe different from the recognition site. Moreover, the term recognitionsite comprises both the nuclease agent binding site and thenick/cleavage site irrespective whether the nick/cleavage site is withinor outside the nuclease agent binding site. In another variation, thecleavage by the nuclease agent can occur at nucleotide positionsimmediately opposite each other to produce a blunt end cut or, in othercases, the incisions can be staggered to produce single-strandedoverhangs, also called “sticky ends”, which can be either 5′ overhangs,or 3′ overhangs.

Any nuclease agent that induces a nick or double-strand break into adesired recognition site can be used in the methods and compositionsdisclosed herein. A naturally-occurring or native nuclease agent can beemployed so long as the nuclease agent induces a nick or double-strandbreak in a desired recognition site. Alternatively, a modified orengineered nuclease agent can be employed. An “engineered nucleaseagent” comprises a nuclease that is engineered (modified or derived)from its native form to specifically recognize and induce a nick ordouble-strand break in the desired recognition site. Thus, an engineerednuclease agent can be derived from a native, naturally-occurringnuclease agent or it can be artificially created or synthesized. Themodification of the nuclease agent can be as little as one amino acid ina protein cleavage agent or one nucleotide in a nucleic acid cleavageagent. In some embodiments, the engineered nuclease induces a nick ordouble-strand break in a recognition site, wherein the recognition sitewas not a sequence that would have been recognized by a native(non-engineered or non-modified) nuclease agent. Producing a nick ordouble-strand break in a recognition site or other DNA can be referredto herein as “cutting” or “cleaving” the recognition site or other DNA.

Active variants and fragments of the exemplified recognition sites arealso provided. Such active variants can comprise at least 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the given recognition site, wherein the activevariants retain biological activity and hence are capable of beingrecognized and cleaved by a nuclease agent in a sequence-specificmanner. Assays to measure the double-strand break of a recognition siteby a nuclease agent are known in the art and generally measure theability of a nuclease to cut the recognition site.

The recognition site of the nuclease agent can be positioned anywhere inor near the target locus. The recognition site can be located within acoding region of a gene, or within regulatory regions, which influenceexpression of the gene. Thus, a recognition site of the nuclease agentcan be located in an intron, an exon, a promoter, an enhancer, aregulatory region, or any non-protein coding region.

In one embodiment, the nuclease agent is a Transcription Activator-LikeEffector Nuclease (TALEN). TAL effector nucleases are a class ofsequence-specific nucleases that can be used to make double-strandbreaks at specific target sequences in the genome of a prokaryotic oreukaryotic organism. TAL effector nucleases are created by fusing anative or engineered transcription activator-like (TAL) effector, orfunctional part thereof, to the catalytic domain of an endonuclease,such as, for example, FokI. The unique, modular TAL effector DNA bindingdomain allows for the design of proteins with potentially any given DNArecognition specificity. Thus, the DNA binding domains of the TALeffector nucleases can be engineered to recognize specific DNA targetsites and thus, used to make double-strand breaks at desired targetsequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS10.1073/pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432;Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc.Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al. (2011)Nature Biotechnology 29:143-148; all of which are herein incorporated byreference.

Examples of suitable TAL nucleases, and methods for preparing suitableTAL nucleases, are disclosed, e.g., in US Patent Application No.2011/0239315 A1, 2011/0269234 A1, 2011/0145940 A1, 2003/0232410 A1,2005/0208489 A1, 2005/0026157 A1, 2005/0064474 A1, 2006/0188987 A1, and2006/0063231 A1 (each hereby incorporated by reference). In variousembodiments, TAL effector nucleases are engineered that cut in or near atarget nucleic acid sequence in, e.g., a genomic locus of interest,wherein the target nucleic acid sequence is at or near a sequence to bemodified by a targeting vector. The TAL nucleases suitable for use withthe various methods and compositions provided herein include those thatare specifically designed to bind at or near target nucleic acidsequences to be modified by targeting vectors as described herein.

In one embodiment, each monomer of the TALEN comprises 12-25 TALrepeats, wherein each TAL repeat binds a lbp subsite. In one embodiment,the nuclease agent is a chimeric protein comprising a TAL repeat-basedDNA binding domain operably linked to an independent nuclease. In oneembodiment, the independent nuclease is a FokI endonuclease. In oneembodiment, the nuclease agent comprises a first TAL-repeat-based DNAbinding domain and a second TAL-repeat-based DNA binding domain, whereineach of the first and the second TAL-repeat-based DNA binding domain isoperably linked to a FokI nuclease, wherein the first and the secondTAL-repeat-based DNA binding domain recognize two contiguous target DNAsequences in each strand of the target DNA sequence separated by about 6bp to about 40 bp cleavage site, and wherein the FokI nucleases dimerizeand make a double strand break at a target sequence.

In one embodiment, the nuclease agent comprises a first TAL-repeat-basedDNA binding domain and a second TAL-repeat-based DNA binding domain,wherein each of the first and the second TAL-repeat-based DNA bindingdomain is operably linked to a FokI nuclease, wherein the first and thesecond TAL-repeat-based DNA binding domain recognize two contiguoustarget DNA sequences in each strand of the target DNA sequence separatedby a 5 bp or 6 bp cleavage site, and wherein the FokI nucleases dimerizeand make a double strand break.

The nuclease agent employed in the various methods and compositionsdisclosed herein can further comprise a zinc-finger nuclease (ZFN). Inone embodiment, each monomer of the ZFN comprises 3 or more zincfinger-based DNA binding domains, wherein each zinc finger-based DNAbinding domain binds to a 3 bp subsite. In other embodiments, the ZFN isa chimeric protein comprising a zinc finger-based DNA binding domainoperably linked to an independent nuclease. In one embodiment, theindependent endonuclease is a FokI endonuclease. In one embodiment, thenuclease agent comprises a first ZFN and a second ZFN, wherein each ofthe first ZFN and the second ZFN is operably linked to a FokI nuclease,wherein the first and the second ZFN recognize two contiguous target DNAsequences in each strand of the target DNA sequence separated by about 6bp to about 40 bp cleavage site or about a 5 bp to about 6 bp cleavagesite, and wherein the FokI nucleases dimerize and make a double strandbreak. See, for example, US20060246567; US20080182332; US20020081614;US20030021776; WO/2002/057308A2; US20130123484; US20100291048; and,WO/2011/017293A2, each of which is herein incorporated by reference.

In one embodiment of the methods provided herein, the nuclease agentcomprises (a) a chimeric protein comprising a zinc finger-based DNAbinding domain fused to a FokI endonuclease; or, (b) a chimeric proteincomprising a Transcription Activator-Like Effector Nuclease (TALEN)fused to a FokI endonuclease.

In still another embodiment, the nuclease agent is a meganuclease.Meganucleases have been classified into four families based on conservedsequence motifs, the families are the LAGLIDADG (SEQ ID NO: 16),GIY-YIG, H—N—H, and His-Cys box families. These motifs participate inthe coordination of metal ions and hydrolysis of phosphodiester bonds.HEases are notable for their long recognition sites, and for toleratingsome sequence polymorphisms in their DNA substrates. Meganucleasedomains, structure and function are known, see for example, Guhan andMuniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al.,(2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell MolLife Sci 55:1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moureet al., (2002) Nat Struct Biol 9:764. In some examples a naturallyoccurring variant, and/or engineered derivative meganuclease is used.Methods for modifying the kinetics, cofactor interactions, expression,optimal conditions, and/or recognition site specificity, and screeningfor activity are known, see for example, Epinat et al., (2003) NucleicAcids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905;Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002)Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mot Biol342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames etal., (2005) Nucleic Acids Res 33:e178; Smith et al., (2006) NucleicAcids Res 34:e149; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chenand Zhao, (2005) Nucleic Acids Res 33:e154; WO2005105989; WO2003078619;WO2006097854; WO2006097853; WO2006097784; and WO2004031346.

Any meganuclease can be used herein, including, but not limited to,I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI,I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP,I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII,I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI,I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP,I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI,I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP,I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP,I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I,I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII,I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI,PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII,PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI,PI-TliI, PI-TliII, or any active variants or fragments thereof.

In one embodiment, the meganuclease recognizes double-stranded DNAsequences of 12 to 40 base pairs. In one embodiment, the meganucleaserecognizes one perfectly matched target sequence in the genome. In oneembodiment, the meganuclease is a homing nuclease. In one embodiment,the homing nuclease is a LAGLIDADG (SEQ ID NO: 16) family of homingnuclease. In one embodiment, the LAGLIDADG (SEQ ID NO: 16) family ofhoming nuclease is selected from I-SceI, I-CreI, and I-Dmol.

Nuclease agents can further comprise restriction endonucleases, whichinclude Type I, Type II, Type III, and Type IV endonucleases. Type I andType III restriction endonucleases recognize specific recognition sites,but typically cleave at a variable position from the nuclease bindingsite, which can be hundreds of base pairs away from the cleavage site(recognition site). In Type II systems the restriction activity isindependent of any methylase activity, and cleavage typically occurs atspecific sites within or near to the binding site. Most Type II enzymescut palindromic sequences, however Type Ha enzymes recognizenon-palindromic recognition sites and cleave outside of the recognitionsite, Type IIb enzymes cut sequences twice with both sites outside ofthe recognition site, and Type IIs enzymes recognize an asymmetricrecognition site and cleave on one side and at a defined distance ofabout 1-20 nucleotides from the recognition site. Type IV restrictionenzymes target methylated DNA. Restriction enzymes are further describedand classified, for example in the REBASE database (webpage atrebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-20),Roberts et al., (2003) Nucleic Acids Res 31:1805-12, and Belfort et al.,(2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press,Washington, D.C.).

The nuclease agent employed in the various methods and compositions canalso comprise a CRISPR/Cas system. Such systems can employ, for example,a Cas9 nuclease, which in some instances, is codon-optimized for thedesired cell type in which it is to be expressed. The system furtheremploys a fused crRNA-tracrRNA construct that functions with thecodon-optimized Cas9. This single RNA is often referred to as a guideRNA or gRNA. Within a gRNA, the crRNA portion is identified as the‘target sequence’ for the given recognition site and the tracrRNA isoften referred to as the ‘scaffold’. Briefly, a short DNA fragmentcontaining the target sequence is inserted into a guide RNA expressionplasmid. The gRNA expression plasmid comprises the target sequence (insome embodiments around 20 nucleotides), a form of the tracrRNA sequence(the scaffold) as well as a suitable promoter that is active in the celland necessary elements for proper processing in eukaryotic cells. Manyof the systems rely on custom, complementary oligos that are annealed toform a double stranded DNA and then cloned into the gRNA expressionplasmid. The gRNA expression cassette and the Cas9 expression cassetteis then introduced into the cell. See, for example, Mali P et al. (2013)Science 2013 Feb. 15; 339(6121):823-6; Jinek M et al. Science 2012 Aug.17; 337(6096):816-21; Hwang W Y et al. Nat Biotechnol 2013 March;31(3):227-9; Jiang W et al. Nat Biotechnol 2013 March; 31(3):233-9; and,Cong L et al. Science 2013 Feb. 15; 339(6121):819-23, each of which isherein incorporated by reference.

In one embodiment, the method for modifying a genomic locus of interestin a pluripotent rat cell further comprises introducing into thepluripotent rat cell: (a) a first expression construct comprising afirst promoter operably linked to a first nucleic acid sequence encodinga Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)-associated (Cas) protein; (b) a second expression constructcomprising a second promoter operably linked to a genomic targetsequence linked to a guide RNA (gRNA), wherein the genomic targetsequence is flanked on the 3′ end by a Protospacer Adjacent Motif (PAM)sequence. In one embodiment, the genomic target sequence comprises thenucleotide sequence of GNNNNNNNNNNNNNNNNNNNNGG (GN₁₋₂₀GG; SEQ ID NO: 1).In one embodiment, the genomic target sequence comprises SEQ ID NO:23,wherein N is between 1 and 20 nucleotides in length. In anotherembodiment, the genomic target sequence comprises between 14 and 20nucleotides in length of SEQ ID NO:1.

In one embodiment, the gRNA comprises a third nucleic acid sequenceencoding a Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). Inspecific embodiments, the Cas protein is Cas9.

In some embodiments, the gRNA comprises (a) the chimeric RNA of thenucleic acid sequence 5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′ (SEQ ID NO: 2);or, (b) the chimeric RNA of the nucleic acid sequence5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCG-3′ (SEQ NO: 3).

In another embodiment, the crRNA comprises5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU-3′ (SEQ ID NO: 4);5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAG (SEQ ID NO: 5); or5′-GAGUCCGAGCAGAAGAAGAAGUUUUA-3′ (SEQ ID NO: 6).

In yet other embodiments, the tracrRNA comprises. 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 7) or 5′-AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3′ (SEQ ID NO: 8).

In one embodiment, the Cas protein is a type I Cas protein. In oneembodiment, the Cas protein is a type II Cas protein. In one embodiment,the type II Cas protein is Cas9. In one embodiment, the first nucleicacid sequence encodes a human codon-optimized Cas protein.

In one embodiment, the first nucleic acid comprises a mutation thatdisrupts at least one amino acid residue of nuclease active sites in theCas protein, wherein the mutant Cas protein generates a break in onlyone strand of the target DNA region, and wherein the mutation diminishesnonhomologous recombination in the target DNA region.

In one embodiment, the first nucleic acid that encodes the Cas proteinfurther comprises a nuclear localization signal (NLS). In oneembodiment, the nuclear localization signal is a SV40 nuclearlocalization signal.

In one embodiment, the second promoter that drives the expression of thegenomic target sequence and the guide RNA (gRNA) is an RNA polymeraseIII promoter. In one embodiment, the RNA polymerase III promoter is ahuman U6 promoter. In one embodiment, the RNA polymerase III promoter isa rat U6 polymerase III promoter. In one embodiment, the RNA polymeraseIII promoter is a mouse U6 polymerase III promoter.

In one embodiment, the nucleic acid sequences encoding crRNA and thetracrRNA are linked via a synthetic loop, wherein, upon expression, thecrRNA and the tracrRNA forms a crRNA:tracrRNA duplex.

In one embodiment, the first expression construct and the secondexpression construct are expressed from a same plasmid.

In one embodiment, the first and the second expression constructs areintroduced together with the LTVEC. In one embodiment, the first and thesecond expression constructs are introduced separately from the LTVECover a period of time.

In one embodiment, the method comprises introducing a plurality of thesecond construct and a plurality of the LTVEC for multiplex editing ofdistinct target loci as described herein.

Active variants and fragments of nuclease agents (i.e. an engineerednuclease agent) are also provided. Such active variants can comprise atleast 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to the native nuclease agent, whereinthe active variants retain the ability to cut at a desired recognitionsite and hence retain nick or double-strand-break-inducing activity. Forexample, any of the nuclease agents described herein can be modifiedfrom a native endonuclease sequence and designed to recognize and inducea nick or double-strand break at a recognition site that was notrecognized by the native nuclease agent. Thus in some embodiments, theengineered nuclease has a specificity to induce a nick or double-strandbreak at a recognition site that is different from the correspondingnative nuclease agent recognition site. Assays for nick ordouble-strand-break-inducing activity are known and generally measurethe overall activity and specificity of the endonuclease on DNAsubstrates containing the recognition site.

The nuclease agent may be introduced into the cell by any means known inthe art. The polypeptide encoding the nuclease agent may be directlyintroduced into the cell. Alternatively, a polynucleotide encoding thenuclease agent can be introduced into the cell. When a polynucleotideencoding the nuclease agent is introduced into the cell, the nucleaseagent can be transiently, conditionally or constitutively expressedwithin the cell. Thus, the polynucleotide encoding the nuclease agentcan be contained in an expression cassette and be operably linked to aconditional promoter, an inducible promoter, a constitutive promoter, ora tissue-specific promoter. Such promoters of interest are discussed infurther detail elsewhere herein. Alternatively, the nuclease agent isintroduced into the cell as an mRNA encoding or comprising a nucleaseagent.

In one embodiment, the crRNA and the tracrRNA are expressed as separateRNA transcripts.

In specific embodiments, the polynucleotide encoding the nuclease agentis stably integrated in the genome of the cell and operably linked to apromoter active in the cell. In other embodiments, the polynucleotideencoding the nuclease agent is in the same targeting vector comprisingthe insert nucleic acid, while in other instances the polynucleotideencoding the nuclease agent is in a vector or a plasmid that is separatefrom the targeting vector comprising the insert nucleic acid.

When the nuclease agent is provided to the cell through the introductionof a polynucleotide encoding the nuclease agent, such a polynucleotideencoding a nuclease agent can be modified to substitute codons having ahigher frequency of usage in the cell of interest, as compared to thenaturally occurring polynucleotide sequence encoding the nuclease agent.For example the polynucleotide encoding the nuclease agent can bemodified to substitute codons having a higher frequency of usage in agiven prokaryotic or eukaryotic cell of interest, including a bacterialcell, a yeast cell, a human cell, a non-human cell, a mammalian cell, arodent cell, a mouse cell, a rat cell or any other host cell ofinterest, as compared to the naturally occurring polynucleotidesequence.

In one embodiment, the endonuclease agent is introduced together withthe LTVEC. In one embodiment, the endonuclease agent is introducedseparately from the LTVEC over a period of time. In one embodiment, theendonuclease agent is introduced prior to the introduction of the LTVEC.In one embodiment, the endonuclease agent is introduced into the rat EScell following introduction of the LTVEC.

In one embodiment, the endonuclease agent is an expression constructcomprising a nucleic acid sequence encoding an endonuclease, wherein thenucleic acid sequence is operably linked to a promoter. In oneembodiment, the promoter is a constitutively active promoter. In oneembodiment, the promoter is an inducible promoter. In one embodiment,the promoter is active in the pluripotent rat cell. In one embodiment,the endonuclease agent is an mRNA encoding an endonuclease.

B. Methods for Integrating a Polynucleotide of Interest into a TargetLocus

Methods for modifying a target locus of interest are provided. In oneembodiment, a target locus in a pluripotent rat cell is targeted forgenetic modification. Such a method comprises: (a) introducing into thepluripotent rat cell a targeting vector comprising an insert nucleicacid flanked with a 5′ rat homology arm and a 3′ rat homology arm; and(b) identifying a genetically modified pluripotent rat cell comprisingthe targeted genetic modification at the target locus, wherein thetargeted genetic modification is capable of being transmitted throughthe germline. In specific embodiments, the sum total of the 5′ homologyarm and the 3′ homology arm is at east 10 kb and/or a large targetingvector is employed.

In other embodiments, the size of the sum total of the total of the 5′and 3′ homology arms of the LTVEC is about 10 kb to about 150 kb, about10 kb to about 100 kb, about 10 kb to about 75 kb, about 20 kb to about150 kb, about 20 kb to about 100 kb, about 20 kb to about 75 kb, about30 kb to about 150 kb, about 30 kb to about 100 kb, about 30 kb to about75 kb, about 40 kb to about 150 kb, about 40 kb to about 100 kb, about40 kb to about 75 kb, about 50 kb to about 150 kb, about 50 kb to about100 kb, or about 50 kb to about 75 kb, about 10 kb to about 30 kb, about20 kb to about 40 kb, about 40 kb to about 60 kb, about 60 kb to about80 kb, about 80 kb to about 100 kb, about 100 kb to about 120 kb, orfrom about 120 kb to about 150 kb. In one embodiment, the size of thedeletion is the same or similar to the size of the sum total of the 5′and 3′ homology arms of the LTVEC.

The pluripotent rat cell can be a rat embryonic stem cell. In a specificembodiment, (a) the rat ES cell is derived from a DA strain or an ACIstrain; or, (b) the rat ES cell is characterized by expression of apluripotency marker comprising Oct-4, Sox-2, alkaline phosphatase, or acombination thereof. In other instances, the rat embryonic stem cellemployed comprises a rat ES cell as described in US 2014-0235933, hereinincorporated by reference in its entirety.

As described elsewhere herein, the insert nucleic acid can be anynucleic acid sequence. In non-limiting embodiments, (a) the insertnucleic acid comprises a replacement of an endogenous rat nucleic acidsequence with a homologous or a orthologous mammalian nucleic acidsequence; (b) the insert nucleic acid comprises a deletion of anendogenous rat nucleic acid sequence; (c) the insert nucleic acidcomprises a deletion of an endogenous rat nucleic acid sequence, whereinthe deletion ranges from 5 kb to 200 kb or from 5 kb to 3 Mb (asdiscussed in detail elsewhere herein); (d) the insert nucleic acidcomprises an addition of an exogenous nucleic acid sequence (includingfor example an exogenous nucleic acid sequence ranging from about 5 kbto about 10 kb, from about 10 kb to about 20 kb, from about 20 kb toabout 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150kb, from about 150 kb to about 200 kb, from about 200 kb to about 250kb, from about 250 kb to about 300 kb, from about 300 kb to about 350kb, or from about 350 kb to about 400 kb); (e) the insert nucleic addcomprises an exogenous nucleic acid sequence comprising a homologous oran orthologous nucleic acid sequence; (f) the homologous or theorthologous nucleic acid sequence of (a) wherein the nucleic acidsequence is a human nucleic acid sequence; (g) the insert nucleic acidcomprises the homologous or the orthologous nucleic acid sequence of (a)wherein the nucleic acid sequence is a chimeric nucleic acid sequencecomprising a human and a rat nucleic acid sequence; (h) the insertnucleic acid comprises the exogenous nucleic acid sequence of (e),wherein the insert nucleic acid ranges from about 5 kb to about 200 kb;(i) the insert nucleic acid comprises a conditional allele flanked withsite-specific recombinase target sequences; (j) the insert nucleic acidcomprises a reporter gene operably linked to a promoter; (k) the insertnucleic acid comprises one or more unrearranged human immunoglobulinheavy chain V_(H) gene segments, one or more unrearranged humanimmunoglobulin heavy chain D gene segments, and one or more unrearrangedhuman immunoglobulin heavy chain J_(H) gene segments, which are operablylinked to a rodent heavy chain constant region nucleic acid sequence;(l) the insert nucleic acid comprises a rearranged human immunoglobulinheavy chain variable region nucleic acid sequence operably linked to arodent heavy chain constant region nucleic acid sequence; (m) the insertnucleic acid comprises one or more unrearranged human immunoglobulinV_(κ) or V_(λ) gene segments and one or more unrearranged humanimmunoglobulin J_(κ) or J_(λ) gene segments, which are operably linkedto a mammalian immunoglobulin λ or κ light chain light chain constantregion nucleic acid sequence; (n) the insert nucleic acid comprises arearranged human immunoglobulin λ or κ light chain variable regionnucleic acid sequence operably linked to a mammalian immunoglobulin λ orκ light chain light chain constant region nucleic acid sequence; (o) themammalian heavy chain constant region nucleic acid sequence of (k)and/or (l) comprises a rat constant region nucleic acid sequence, ahuman constant region nucleic acid sequence, or a combination thereof;or, (p) the mammalian immunoglobulin λ or κ light chain constant regionnucleic acid of (m) and/or (n) comprises a rat constant region nucleicacid sequence, a human constant region nucleic acid sequence, or acombination thereof.

In one embodiment, the insert nucleic acid comprises one or morefunctional human V_(H) gene segments comprising V_(H)1-2, V_(H)1-3,V_(H)1-8, V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58,V_(H)1-69, V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9,V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21,V_(H)3-23, V_(H)3-30, V_(H)3-30-3, V_(H)3-30-5, V_(H)3- 33, V_(H)3-35,V_(H)3-38, V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64,V_(H)3-66, V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28,V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4- 39,V_(H)4-59, V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, or acombination thereof.

In one embodiment, the insert nucleic acid comprises one or morefunctional human D gene segments comprising D1-1, D1-7, D1-14, D1-20,D1-26, D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4,D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6, D6-13, D6-19,D6-25, D7-27, or a combination thereof.

In one embodiment, the insert nucleic acid comprises one or morefunctional J_(H) gene segments comprising J_(H)1, J_(H)2, J_(H)3,J_(H)4, J_(H)5, J_(H)6, or a combination thereof. In one embodiment, theinsert nucleic acid comprises one or more human Vκ gene segmentscomprising Vκ4-1, Vκ7-3, Vκ2-4, Vκ1-5, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13,Vκ2-14, Vκ3-15, Vκ1-16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22,Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31,Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40,or a combination thereof.

In one embodiment, the insert nucleic acid comprises one or more humanVλ gene segments comprising Vλ3-1, Vλ2-8, Vλ3-10, Vλ2-11, Vλ3-12,Vλ2-14, Vλ2-16, Vλ2-18, Vλ3-19, Vλ3-21, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27or a combination thereof.

In one embodiment, the insert nucleic acid comprises one or more humanJκ gene segments comprising Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combinationthereof.

In specific embodiments, upon modification of the target locus in apluripotent rat cell, the genetic modification is transmitted throughthe germline.

In one embodiment, the insert nucleic acid sequence comprises apolynucleotide that when integrated into the genome will produce agenetic modification of a region of the rat ApoE locus, wherein thegenetic modification at the ApoE locus results in a decrease in ApoEactivity, an increase in ApoE activity or a modulation of ApoE activity.In one embodiment, an ApoE knockout is generated.

In one embodiment, the insert nucleic acid sequence comprises apolynucleotide that when integrated into the genome will produce agenetic modification of a region of the rat interleukin-2 receptor gammalocus, wherein the genetic modification at the interleukin-2 receptorgamma locus results in a decrease in interleukin-2 receptor activity, anincrease in interleukin-2 receptor gamma activity, or a modulation ofinterleukin-2 receptor activity. In one embodiment, an interleukin-2receptor knockout is generated.

In still another embodiment, the insert nucleic acid sequence comprisesa polynucleotide that when integrated into the genome will produce agenetic modification of a region of the rat Rag1 locus, the rat Rag2locus and/or the rat Rag2/Rag1 locus, wherein the genetic modificationat the rat Rag1, Rag2 and/or Rag2/Rag1 locus results in a decrease in inRag1, Rag2 or Rag1 and Rag2 protein activity, an increase in Rag1, Rag2or Rag1 and Rag2 protein activity, or a modulation in Rag1, Rag2 or Rag1 and Rag2 protein activity. In one embodiment, a Rag1, Rag2 orRag2/Rag1 knockout is generated.

In further embodiments, the insert nucleic acid results in thereplacement of a portion of the rat ApoE locus, the interleukin-2receptor gamma locus and/or Rag2 locus, and/or Rag1 locus and/orRag2/Rag1 locus with the corresponding orthologous portion of an ApoElocus, an interleukin-2 receptor gamma locus, a Rag2 locus, a Rag1 locusand/or a Rag2/Rag1 locus from another organism.

Still other embodiments, the insert nucleic acid comprises apolynucleotide sharing across its full length least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to a portion of an ApoE locus, aninterleukin-2 receptor gamma locus, a Rag2 locus, a Rag1 locus and/or aRag2/Rag1 locus it is replacing.

The given insert polynucleotide and the corresponding region of the ratlocus being replaced can be a coding region, an intron, an exon, anuntranslated region, a regulatory region, a promoter, or an enhancer orany combination thereof. Moreover, the given insert polynucleotideand/or the region of the rat locus being replaced can be of any desiredlength, including for example, between 10-100 nucleotides in length,100-500 nucleotides in length, 500-1 kb nucleotide in length, 1 Kb to1.5 kb nucleotide in length, 1.5 kb to 2 kb nucleotides in length, 2 kbto 2.5 kb nucleotides in length, 2.5 kb to 3 kb nucleotides in length, 3kb to 5 kb nucleotides in length, 5 kb to 8 kb nucleotides in length, 8kb to 10 kb nucleotides in length or more. In other instances, the sizeof the insertion or replacement is from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, from about 350 kb toabout 400 kb, from about 400 kb to about 800 kb, from about 800 kb to 1Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb,from about 2 Mb, to about 2.5 Mb, from about 2.5 Mb to about 2.8 Mb,from about 2.8 Mb to about 3 Mb. In other embodiments, the given insertpolynucleotide and/or the region of the rat locus being replaced is atleast 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides or atleast 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb or greater.

i. Methods for Modifying a Target Locus of a Rat Nucleic Acid ViaBacterial Homologous Recombination (BHR)

Methods and compositions are provided for modifying a target locus of arat nucleic acid via bacterial homologous recombination (BHR) in aprokaryotic cell. Such methods find use in utilizing bacterialhomologous recombination in a prokaryotic cell to genetically modify atarget locus of a rat nucleic acid in order to create a targetingvector. Such a targeting vector comprising the genetically modifiedtarget locus can be introduced into a eukaryotic cell, for example, apluripotent rat cell. “Homologous recombination” includes the exchangeof DNA fragments between two DNA molecules at cross-over sites withinregions of homology. Thus, “bacterial homologous recombination” or “BHR”includes homologous recombination that occurs in bacteria.

Methods for modifying a target locus of a rat nucleic acid via bacterialhomologous recombination (BHR) are provided that comprise introducinginto a prokaryotic cell a targeting vector comprising an insert nucleicacid flanked with a 5′ rat homology arm and a 3′ rat homology arm,wherein the prokaryotic cell comprises a rat nucleic acid and is capableof expressing a recombinase that mediates the BHR at the target locus.Such targeting vectors can include any of the large targeting vectorsdescribed herein.

In one embodiment, the method comprises introducing into a prokaryoticcell: (i) a first construct comprising a rat nucleic acid having a DNAsequence of interest; (ii) a second targeting construct comprising aninsert nucleic acid flanked with a rat 5′ homology arm and a rat 3′homology arm, and (iii) a third construct encoding a recombinase thatmediates bacterial homologous recombination. In one embodiment, thefirst, the second, and the third construct are introduced into theprokaryotic cell separately over a period of time. In one embodiment,the prokaryotic cell comprises a nucleic acid that encodes therecombinase, and the method does not require introduction of the thirdconstruct. In one embodiment, the recombinase is expressed under thecontrol of an inducible promoter.

In one embodiment the first construct comprising the rat nucleic acid isderived from a bacterial artificial chromosome (BAC) or yeast artificialchromosome (YAC).

A prokaryotic cell comprising the insert nucleic acid at the targetgenomic locus can be selected. This method can be serially repeated asdisclosed herein to allow the introduction of multiple insert nucleicacids at the targeted rat locus in the prokaryotic cell. Once the targetrat nucleic acid locus is “built” within the prokaryotic cell, atargeting vector comprising the modified rat target locus can beisolated from the prokaryotic cell and introduced into a target genomiclocus within a mammalian cell (i.e., a rat cell, a pluripotent rat cell,or a rat embryonic stem cell).

Preferred rat cells for receiving targeting vectors are described in US2014-0235933, the contents of which are summarized herein. These ratcells are pluripotent rat cells capable of sustaining their pluripotencyfollowing one or more targeted genetic modifications in vitro, and arecapable of transmitting the targeted genetic modifications through thegermline.

Electroporated pluripotent rat cells are plated at a high density forthe selection of drug-resistant cells comprising the targeting vector.The drug selection process removes the majority of the plated cells(˜99%), leaving behind individual colonies, each of which is a clonederived from a single cell. Of the remaining cells, most cells(˜80-100%) contain the targeting vector (comprising a drug selectioncassette) integrated at a random location in the genome. Therefore, thecolonies are picked individually and genotyped to identify rat ES cellsharboring the targeting vector at the correct genomic location (e.g.,using the modification of allele assay described below).

A high-throughput quantitative assay, namely, modification of allele(MOA) assay, can be used for genotyping. Such an assay allows alarge-scale screening of a modified allele(s) in a parental chromosomefollowing a genetic modification. The MOA assay can be carried out viavarious analytical techniques, including, but not limited to, aquantitative PCR, e.g., a real-time PCR (qPCR). For example, thereal-time PCR comprises a first primer set that recognizes the targetlocus and a second primer set that recognizes a non-targeted referencelocus. In addition, the primer set comprises a fluorescent probe thatrecognizes the amplified sequence. In one embodiment, the quantitativeassay is carried out via Invader Probes®. In one embodiment, thequantitative assay is carried out via MMP Assays®. In one embodiment,the quantitative assay is carried out via TaqMan® Molecular Beacon. Inone embodiment, the quantitative assay is carried out via Eclipse™ probetechnology. (See, for example, US2005/0144655, which is incorporated byreference herein in its entirety).

The selected pluripotent rat cell or the rat ES cells comprising thetargeted genetic modification can then be introduced into a host ratembryo, for example, a pre-morula stage or blastocyst stage rat embryo,and implanted in the uterus of a surrogate mother to generate a founderrat (F0 rat). Subsequently, the founder rat can be bred to a wild-typerat to create F1 progeny heterozygous for the genetic modification.Mating of the heterozygous F1 rat can produce progeny homozygous for thegenetic modification. Mating of the heterozygous F1 rat can produceprogeny homozygous for the genetic modification. In some embodiments,various genetic modifications of the target loci described herein can becarried out using a large targeting vector (LTVEC) as described indetail elsewhere herein. For example, an LTVEC can be derived fromBacterial Artificial Chromosome (BAC) DNA using VELOCIGENE® geneticengineering technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela, D. M. et al. (2003), High-throughput engineering of themouse genome coupled with high-resolution expression analysis, NatureBiotechnology 21(6): 652-659, which is incorporated herein by referencein their entireties).

Use of bacterial homologous recombination (BHR) to generate a largetargeting vector (LTVEC) circumvents the limitations of plasmids inaccommodating a large genomic DNA fragment and consequent low efficiencyof introducing a targeted modification into an endogenous locus inpluripotent rat cells. One or more targeted genetic modifications can beperformed in generating a LTVEC. An exemplary LTVEC produced in theprokaryotic cell can comprises an insert nucleic acid that carries a ratgenomic sequence with one or more genetic modifications or an exogenousnucleic acid (e.g., a homolog or ortholog of a rat nucleic acid), whichis flanked by rat homologous arms complementary to specific genomicregions.

Host prokaryotic cells comprising the various targeting vectorsdescribed herein are also provided. Such prokaryotic cells include, butare not limited to, bacteria such as E. coli. In one embodiment, a hostprokaryotic cell comprises a targeting vector comprising an insertnucleic acid flanked with a 5′ rat homology arm and a 3′ rat homologyarm, wherein the insert nucleic acid ranges from about 5 kb to about 200kb.

The host prokaryotic cell can further comprise a nucleic acid thatencodes a recombinase polypeptide or the nucleic acid that encodes therecombinase polypeptide is operably linked to an inducible promoter.

Further provided are various methods and compositions, which employ theLTVEC as described herein in combination with a prokaryotic cell inorder to produce targeted genetic modifications, Such compositions andmethods are discussed elsewhere herein.

Methods for modifying a target locus of a nucleic acid via bacterialhomologous recombination (BHR) are provided that comprise introducinginto a prokaryotic cell a targeting vector comprising an insert nucleicacid flanked with a 5′ homology arm and a 3′ homology arm, wherein theprokaryotic cell comprises nucleic acids corresponding to the 5′ and 3′homology arms and the prokaryotic cell is capable of expressing arecombinase that mediates the BHR at the target locus. Such targetingvectors can include any of the large targeting vectors described herein.Such methods can employ a LTVEC as discussed in detail herein andfurther employ the CRISPR/Cas system as discussed elsewhere herein.

ii. Methods for Modifying a Target Locus of Interest in a PluripotentRat Cell

Further provided is a method for modifying a target locus of interest ina pluripotent rat cell via targeted genetic modification, comprising (a)introducing into the pluripotent rat cell a targeting vector comprisingan insert nucleic acid flanked with a 5′ rat homology arm and a 3′ rathomology arm, wherein the sum total of the 5′ homology arm and the 3′homology arm is at least 10 kb; and (b) identifying a geneticallymodified pluripotent rat cell comprising the targeted geneticmodification at the target locus of interest. In one embodiment; the sumtotal of the 5′ homology arm and the 3′ homology arm is at least about16 kb to about 30 kb. In specific embodiments, the targeted geneticmodification is capable of being transmitted through the germline. Suchtargeting vectors can include any of the large targeting vectorsdescribed herein.

In one aspect, a method for modifying a genomic locus of interest in apluripotent rat cell via targeted genetic modification is provided,comprising: (a) providing a pluripotent rat cell that is able to sustainits pluripotency following at least one targeted genetic modification ofits genome and is able to transmit the targeted modification to agermline of an F1 generation; (b) introducing a large targeting vector(LTVEC) into the pluripotent rat cell, wherein the LTVEC comprises aninsert nucleic acid flanked with a 5′ homology arm and a 3′ homologyarm, wherein the 5′ homology arm and the 3′ homology arm comprise a ratgenomic DNA fragment; and (c) identifying a genetically modifiedpluripotent rat cell comprising the targeted genetic modification.

Various methods can be used to identify cells having the insert nucleicacid integrated at the target locus of interest. Insertion of the insertnucleic acid at the target locus of interest results in a “modificationof allele”. The term “modification of allele” and methods for thedetection of the modified allele are discussed in further detailelsewhere herein.

In one aspect, a method for modifying a genomic locus of interest in apluripotent rat cell via endonuclease-mediated gene targeting isprovided, the method comprising: (a) providing an isolated pluripotentrat cell that is able to transmit the genetically modified genome to agermline of an F1 generation; (b) introducing into the pluripotent ratcell an endonuclease agent; wherein the endonuclease agent makes a nickor a double strand break at a target DNA sequence located in the genomiclocus of interest, and wherein the nick or the double strand break atthe target DNA sequence in the rat ES cell induces: (i) non-homologousend joining (NHEJ)-mediated DNA repair of the nick or the double strandbreak, wherein the NHEJ-mediated DNA repair generates a mutant allelecomprising an insertion or a deletion of a nucleic acid sequence at thetarget DNA sequence; or (ii) homologous recombination-mediated DNArepair that results in restoration of a wild-type nucleic acid sequence;and (c) identifying the modified genomic locus of interest.

In one aspect, a method for modifying a genomic locus of interest in anisolated rat embryonic stem cell (ES) via a nuclease agent is provided,comprising: (a) providing an isolated rat ES cell that is able totransmit the targeted genetic modification to a germline of an F1generation; (b) introducing into the rat ES cell: (i) a large targetingvector (LTVEC) comprising an insert nucleic acid flanked with a rat 5′homology arm and a rat 3′ homology arm, wherein the insert is a nucleicacid sequence that is at least 5 kb; and (ii) an endonuclease agent,wherein the endonuclease agent makes a nick or a double strand break ata target DNA sequence located in the genomic locus of interest, andwherein the target sequence is not present in the insert nucleic acid;and (c) identifying the targeted genetic modification in the ratembryonic stem (ES) cell.

In one aspect, a method for modifying a genomic locus of interest in apluripotent rat cell via RNA-guided genome engineering is provided, themethod comprising: (a) providing a pluripotent rat cell that is able totransmit the genetically modified genome to a germline of an F1generation; (b) introducing into the pluripotent rat cell: (i) a firstexpression construct comprising a first promoter operably linked to afirst nucleic acid sequence encoding a Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR)-associated (Cas) protein, (ii) asecond expression construct comprising a second promoter operably linkedto a genomic target sequence linked to a guide RNA (gRNA), wherein thegenomic target sequence is flanked on the 3′end by a ProtospacerAdjacent Motif (PAM) sequence. In one embodiment, the genomic targetsequence comprises the nucleotide sequence of GNNNNNNNNNNNNNNNNNNNNGG(GN₁₋₂₀GG; SEQ ID NO: 1). In one embodiment, the genomic target sequencecomprises SEQ ID NO:1, wherein N is between 14 and 20 nucleotides inlength. In one embodiment, the gRNA comprises a third nucleic acidsequence encoding a Clustered Regularly Interspaced Short PalindromicRepeats (CRISPR) RNA (crRNA) and a fourth nucleic acid sequence encodinga trans-activating CRISPR RNA (tracrRNA). In one embodiment, uponexpression, the Cas protein forms a CRISPR-Cas complex comprising thecrRNA and the tracrRNA, and the CRISPR-Cas complex makes a nick or adouble strand break at a target DNA sequence located in the genomiclocus of interest, and wherein the nick or the double strand break atthe target DNA sequence in the pluripotent rat cell induces: (i)non-homologous end joining (NHEJ)-mediated DNA repair of the nick or thedouble strand break created by the CRISPR-Cas complex, wherein the NHEJgenerates a mutant allele comprising an insertion or a deletion of anucleic acid sequence at the target DNA sequence; or (ii) homologousrecombination-mediated DNA repair that results in restoration of awild-type nucleic acid sequence; and (c) identifying the modified thegenomic locus of interest.

In one embodiment, the pluripotent rat cell is an induced ratpluripotent stem cell (iPS). In one embodiment, the pluripotent rat cellis a developmentally restricted progenitor cell.

The presence of a nick or a double-strand break in the recognition sitewithin the selection marker, in various embodiments, increases theefficiency and/or frequency of recombination between a targeting vector(such as a LTVEC) and the targeted locus of interest. In one embodiment,the recombination is homologous recombination. In another embodiment,the recombination is an insertion by non-homologous end joining. Invarious embodiments, in the presence of the nick or double strand break,targeting efficiency of a targeting vector (such as a LTVEC) at thetarget genomic locus is at least about 2-fold higher, at least about3-fold higher, at least about 4-fold higher than in the absence of thenick or double-strand break (using, e.g., the same targeting vector andthe same homology arms and corresponding target sites at the genomiclocus of interest but in the absence of an added nuclease agent thatmakes the nick or double strand break).

In one embodiment, the targeted genetic modification at the target locusis biallelic. By “biallelic” is meant that both alleles of a genecomprise the targeted genetic modification. In certain embodiments, thecombined use of a targeting vector (including, for example, an LTVEC)with a nuclease agent results in biallelic targeted genetic modificationof the genomic locus of interest in a cell as compared to use of thetargeting vector alone. When the targeting vector is used in conjunctionwith a nuclease agent, biallelic targeting efficiency is increased atleast by two-fold, at least three-fold, at least 4-fold or more ascompared to when the targeting vector is used alone. In furtherembodiments, the biallelic targeting efficiency is at least 0.2%, 0.3%,0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5% or higher.

Compositions are provided which comprise a genetically modified rathaving a targeted genetic modification in the interleukin-2 receptorgamma locus or in the ApoE locus. The various methods and compositionsprovided herein allows for these modified loci to be transmitted throughthe germline.

In specific embodiments, a genetically modified rat or a geneticallymodified pluripotent rat cell comprises a genomic locus having atargeted genetic modification in the interleukin-2 gamma receptor locusor having a targeted genetic modification in the ApoE locus, wherein theinterleukin-2 gamma receptor genomic locus or the ApoE locus comprise:(i) a deletion of at least a portion of the interleukin-2 gamma receptorlocus or at least a portion of the ApoE locus; (ii) an insertion of aheterologous nucleic acid sequence into the ApoE locus or into theinterleukin-2 gamma receptor locus; or (iii) a combination thereof,wherein the genetically modified genomic locus is capable of beingtransmitted through the germline.

Methods are further provided that allow for such genetically modifiedrats and for such genetically modified pluripotent rat cells to be made.Such methods include a method for modifying an ApoE genomic locus or aninterleukin-2 gamma receptor locus in a pluripotent rat cell viatargeted genetic modification. The method comprises (a) introducing intothe pluripotent rat cell a targeting vector comprising an insert nucleicacid flanked with a 5′ rat homology arm to the ApoE locus and a 3′ rathomology arm to the ApoE locus, (b) identifying a genetically modifiedpluripotent rat cell comprising the targeted genetic modification at theApoE genomic locus of interest, wherein the targeted geneticmodification is capable of being transmitted through germline.

Additional methods include (a) introducing into the pluripotent rat cella targeting vector comprising an insert nucleic acid flanked with a 5′rat homology arm to the interleukin-2 receptor gamma locus and a 3′ rathomology arm to the interleukin-2 receptor gamma locus, (b) identifyinga genetically modified pluripotent rat cell comprising the targetedgenetic modification at the interleukin-2 receptor gamma locus, whereinthe targeted genetic modification is capable of being transmittedthrough germline.

iii. Methods of Integrating Multiple Polynucleotides of Interest at theTargeted Locus

The various methods and compositions provided herein allow for thetargeted integration of multiple polynucleotides of interest with agiven target locus. The various methods set forth above can besequentially repeated to allow for the targeted integration of anynumber of insert nucleic acids into a given targeted locus. Thus, thevarious methods provide for the insertion of at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more insertnucleic acids into the target locus. In particular embodiments, suchsequential stacking methods allow for the reconstruction of largegenomic regions from a mammalian cell (i.e., a human, a non-human, arodent, a mouse, a monkey, a rat, a hamster, a domesticated mammal or anagricultural animal) into a targeted locus. In such instances, thetransfer and reconstruction of genomic regions that include both codingand non-coding regions allow for the complexity of a given region to bepreserved by retaining, at least in part, the coding regions, thenon-coding regions and the copy number variations found within thenative genomic region. Thus, the various methods provide, for example,methods to generate “heterologous” or “exogenous” genomic regions withinany mammalian cell or animal of interest, particularly within aprokaryotic host cell or within a pluripotent rat cell or a rat ES cell.In one non-limiting example, a “humanized” genomic region within anon-human animal (i.e., within a rat) is generated.

3. A Humanized Genomic Locus

Provided herein are various methods and compositions comprising ahumanized rat locus. As used herein, by “humanized” genomic locus ismeant a region of a non-human genome comprising at least one humannucleic acid sequence. A “humanized rat locus” comprises a region of ratDNA that has a human DNA sequence inserted therein. The human DNAsequence can be a naturally occurring human DNA sequence or it can bemodified from its native form. In specific embodiments, the human DNAshares at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to a native human sequence. If a human sequence is nota native human sequence it at least has greater sequence identity to anative human sequence than it does to an orthologous rat sequence.Moreover, the human DNA sequence can comprise a cDNA, a region of humangenomic DNA, a non-coding regulatory region, or any portion of a coding,genomic, or regulatory region of the human DNA. The human DNA sequenceinserted into the rat locus can comprise any of the insertpolynucleotides as described elsewhere herein. In specific embodiments,the human DNA sequence is orthologous to the rat target locus, while inother instances, the human DNA sequence is homologous to the rat targetlocus.

In one embodiment, the targeted genetic modification is an insertion ora replacement of an endogenous rat nucleic acid sequence with ahomologous or orthologous human nucleic acid sequence. In oneembodiment, the targeted genetic modification comprises an insertion orreplacement of an endogenous rat nucleic acid sequence with a homologousor orthologous human nucleic acid sequence at an endogenous rat locusthat comprises the corresponding rat nucleic acid sequence.

Methods for making a humanized rat locus (or a rat or rat cellcomprising the humanized rat locus) comprise introducing into the targetlocus comprising a rat nucleic acid a human nucleic acid sequence. Inone embodiment, a method of making a humanized rat is provided. Such amethod comprises (a) modifying a genome of a pluripotent rat cell with atargeting vector comprising an insert nucleic acid that comprises ahuman nucleic acid sequence to form a donor cell; (b) introducing thedonor cell into a host rat embryo; and (c) gestating the host rat embryoin a surrogate mother; wherein the surrogate mother produces a ratprogeny that comprises the human nucleic acid sequence. In specificembodiments, the humanized rat locus is capable of being transmittedthrough the germline. In a further embodiment, the targeting vectorcomprises a large targeting vector (LTVEC) and the insert nucleic acidthat comprises a human nucleic acid sequence is at least 5 kb.

In other methods, the humanized rat locus is made by modifying a targetlocus of a rat nucleic acid via bacterial homologous recombination(BHR). The method comprises introducing into a prokaryotic cell atargeting vector comprising an insert nucleic acid flanked with a 5′ rathomology arm and a 3′ rat homology arm, wherein the insert nucleic acidcomprises a human nucleic acid sequence, and wherein the prokaryoticcell comprises a rat nucleic acid and is capable of expressing arecombinase that mediates the BHR at the target locus.

The humanized rat genomic locus can comprise (a) an insertion of ahomologous or orthologous human nucleic acid sequence; (b) a replacementof an endogenous rat nucleic acid sequence with a homologous ororthologous human nucleic acid sequence; or (c) a combination thereof.In specific embodiments, the humanized rat genomic locus is capable ofbeing transmitted through the germline. In still other embodiments, thehuman orthologous sequence replaces the corresponding sequence found inthe rat.

Any human nucleic acid sequence can be used in the methods andcompositions provided herein. Non-limiting examples of human nucleicacid sequences that can be used in the methods and compositions arediscussed in detail elsewhere herein.

The human nucleic acid sequence for insertion into the rat locus ofinterest can be any size. In one embodiment, the human nucleic acidsequence can be from about 500 nucleotides to about 200 kb, from about500 nucleotides to about 5 kb, from about 5 kb to about 200 kb, fromabout 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about20 kb to about 30 kb, from about 30 kb to about 40 kb, from about 40 kbto about 50 kb, from about 60 kb to about 70 kb, from about 80 kb toabout 90 kb, from about 90 kb to about 100 kb, from about 100 kb toabout 11.0 kb, from about 120 kb to about 130 kb, from about 130 kb toabout 140 kb, from about 140 kb to about 150 kb, from about 150 kb toabout 160 kb, from about 160 kb to about 170 kb, from about 170 kb toabout 180 kb, from about 180 kb to about 190 kb, or from about 190 kb toabout 200 kb. In a specific embodiment, the human nucleic acid sequenceis at least 5 kb.

in one embodiment, a rat genomic locus is provided wherein thehomologous or orthologous human nucleic acid sequence comprises (a) oneor more unrearranged human immunoglobulin heavy chain V_(H) genesegments, one or more unrearranged human immunoglobulin heavy chain Dgene segments, and one or more unrearranged human immunoglobulin heavychain J_(H) gene segments, which are operably linked to a mammalianheavy chain constant region nucleic acid sequence; (b) a rearrangedhuman immunoglobulin heavy chain variable region nucleic acid sequenceoperably linked to a mammalian immunoglobulin heavy chain constantregion nucleic acid sequence; (c) one or more unrearranged humanimmunoglobulin V_(κ) or V_(λ) gene segments and one or more unrearrangedhuman immunoglobulin J_(κ) or J_(λ) gene segments, which are operablylinked to a mammalian, immunoglobulin λ or κ light chain light chainconstant region nucleic acid sequence; or, (d) a rearranged humanimmunoglobulin λ or κ light chain variable region nucleic acid sequenceoperably linked to a mammalian immunoglobulin λ or κ light chain lightchain constant region nucleic acid sequence.

In another embodiment, a rat genomic locus is provided wherein (a) themammalian immunoglobulin heavy chain constant region nucleic acidsequence is a rat constant region nucleic acid sequence, a humanconstant region nucleic acid sequence, or a combination thereof; or, (b)the mammalian immunoglobulin λ or κ Light chain light chain constantregion nucleic acid sequence is a rat constant region nucleic acidsequence, a human constant region nucleic acid sequence, or acombination thereof.

In a specific embodiment, a rat genomic locus is provided wherein theimmunoglobulin heavy chain constant region nucleic acid sequence isselected from or comprises a CH1, a hinge, a CH2, a CH3, and/or acombination thereof.

In one embodiment, the rat genomic locus comprises one or morefunctional human V_(H) gene segments comprising V_(H)1-2, V_(H)1-3,V_(H)1-8, V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58,V_(H)1-69, V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9,V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21,V_(H)3-23, V_(H)3-30, V_(H)3-30-3, V_(H)3-30-5, V_(H)3- 33, V_(H)3-35,V_(H)3-38, V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64,V_(H)3-66, V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28,V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4- 39,V_(H)4-59, V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, or acombination thereof.

In one embodiment, the rat genomic locus comprises one or morefunctional human D gene segments commising D1-1, D1-7, D1-14, D1-20,D1-26, D2-2, D2-8, D2-15, D2-21; D3-3, D3-9, D3-10, D3-16, D3-22, D4-4,D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6, D6-13, D6-19,D6-25, D7-27, or a combination thereof.

In one embodiment, the rat genomic locus comprises one or morefunctional higene segments comprising J_(H)1, J_(H)2, J_(H)3, J_(H)4,J_(H)5, J_(H)6, and/or a combination thereof. In one embodiment, theinsert nucleic acid comprises one or more human Vκ gene segmentscomprises Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1-9,Vκ2-10, Vκ3-11, Vκ2-14, Vκ3-15, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22,Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31,Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39, Vκ2-40,or a combination thereof.

In one embodiment, the rat genomic locus comprises one or more human Vλgene segments comprising Vλ3-1, Vλ4-3, Vλ2-8, Vλ3-10, Vλ2-11, Vλ3-12,Vλ2-14, Vλ3-16, Vλ2-18, Vλ3-22, Vλ2-23, Vλ3-25, Vλ3-27, or a combinationthereof.

In one embodiment, the rat genomic locus comprises one or more human Jκgene segments comprising Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, or a combinationthereof.

In yet another embodiment, the rat genomic locus comprises a humanizedgenomic locus comprising a human interleukin-2 receptor (IL2R) nucleicacid sequence or a variant or a fragment thereof is provided. Inspecific embodiments, the IL2R nucleic acid sequence comprises aninterleukin-2 receptor alpha, an interleukin-2 receptor beta, or aninterleukin-2 receptor gamma nucleic acid sequence or variants orfragments thereof.

In further embodiments, a rat genomic locus comprises a humanizedgenomic locus comprising of a portion of the human ApoE locus, the humaninterleukin-2 receptor gamma locus, the human Rag2 locus, the human Rag1locus and/or the human Rag2/Rag1 locus replacing the correspondinghomologous or orthologous portion of the rat ApoE locus, the ratinterleukin-2 receptor gamma locus, the rat Rag2 locus, the rat Rag1locus and/or the rat Rag2/Rag1 locus. In one embodiment, the ratecto-domain of IL-2Rg is replaced with the ecto-domain of human IL-2Rg,with the remainder of the molecule being from the rat.

In another embodiment, a genetically modified rat comprising a humanizedgenomic locus is provided, Such genetically modified rats comprise (a)an insertion of a homologous or orthologous human nucleic acid sequence;(b) a replacement of rat nucleic acid sequence with a homologous ororthologous human nucleic acid sequence at an endogenous genomic locus;or (c) a combination thereof, wherein the humanized genomic locus iscapable of being transmitted through the germline.

Genetically modified rats comprising any of the various humanizedgenomic loci provided herein and described above are also provided.

4. Polynucleotides of Interest

Any polynucleotide of interest may be contained in the various insertnucleic acids and thereby integrated at the target locus. The methodsdisclosed herein, provide for at least 1, 2, 3, 4, 5, 6 or morepolynucleotides of interest to be integrated into the targeted genomiclocus.

The polynucleotide of interest within the insert nucleic acid whenintegrated at the target genomic locus can introduce one or more geneticmodifications into the cell. The genetic modification can comprise adeletion of an endogenous nucleic acid sequence and/or the addition ofan exogenous or heterologous or orthologous polynucleotide into thetarget genomic locus. In one embodiment, the genetic modificationcomprises a replacement of an endogenous nucleic acid sequence with anexogenous polynucleotide of interest at the target genomic locus. Thus,methods provided herein allow for the generation of a geneticmodification comprising a knockout, a deletion, an insertion, areplacement (“knock-in”), a point mutation, a domain swap, an exon swap,an intron swap, a regulatory sequence swap, a gene swap, or acombination thereof. Such modifications may occur upon integration ofthe first, second, third, fourth, fifth, six, seventh, or any subsequentinsert nucleic acids into the target genomic locus.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can comprise a sequence that is native tothe cell it is introduced into; the polynucleotide of interest can beheterologous to the cell it is introduced to; the polynucleotide ofinterest can be exogenous to the cell it is introduced into; thepolynucleotide of interest can be orthologous to the cell it isintroduced into; or the polynucleotide of interest can be from adifferent species than the cell it is introduced into. As used herein“native” in reference to a sequence inserted at the target locus is asequence that is native to the cell having the target locus or native tothe cell from which the target locus was derived (i.e., from a rat). Asused herein, “heterologous” in reference to a sequence includes asequence that originates from a foreign species, or, if from the samespecies, is substantially different or modified from its native form incomposition and/or genomic locus by deliberate human intervention. Asused herein, “exogenous” in reference to a sequence is a sequence thatoriginates from a foreign species. The polynucleotide of interest can befrom any organism of interest including, but not limited to, non-human,a rodent, a hamster, a mouse, a rat, a human, a monkey, an agriculturalmammal or a non-agricultural mammal. The polynucleotide of interest canfurther comprise a coding region, a non-coding region, a regulatoryregion, or a genomic DNA. Thus, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th,and/or any of the subsequent insert nucleic acids can comprise suchsequences.

In one embodiment, the polynucleotide of interest within the insertnucleic acid and/or integrated at the target locus is native to a mousenucleic acid sequence, a human nucleic acid, a non-human nucleic acid, arodent nucleic acid, a rat nucleic acid, a hamster nucleic acid, amonkey nucleic acid, an agricultural mammal nucleic acid, or anon-agricultural mammal nucleic acid. In still further embodiments, thepolynucleotide of interest integrated at the target locus is a fragmentof a genomic nucleic acid. In one embodiment, the genomic nucleic acidis a mouse genomic nucleic acid, a human genomic nucleic acid, anon-human nucleic acid, a rodent nucleic acid, a rat nucleic acid, ahamster nucleic acid, a monkey nucleic acid, an agricultural mammalnucleic acid or a non-agricultural mammal nucleic acid or a combinationthereof.

In one embodiment, the polynucleotide of interest can range from about500 nucleotides to about 200 kb as described above. The polynucleotideof interest can be from about 500 nucleotides to about 5 kb, from about5 kb to about 200 kb, from about 5 kb to about 10 kb, from about 10 kbto about 20 kb, from about 20 kb to about 30 kb, from about 30 kb toabout 40 kb, from about 40 kb to about 50 kb, from about 60 kb to about70 kb, from about 80 kb to about 90 kb, from about 90 kb to about 1.00kb, from about 100 kb to about 110 kb, from about 120 kb to about 130kb, from about 130 kb to about 140 kb, from about 140 kb to about 150kb, from about 150 kb to about 160 kb, from about 160 kb to about 170kb, from about 170 kb to about 180 kb, from about 180 kb to about 190kb, or from about 190 kb to about 200 kb, from about 5 kb to about 10kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb,from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, fromabout 80 kb to about 100 kb, from about 100 kb to about 150 kb, fromabout 150 kb to about 200 kb, from about 200 kb to about 250 kb, fromabout 250 kb to about 300 kb, from about 300 kb to about 350 kb, or fromabout 350 kb to about 400 kb.

The polynucleotide of interest within the insert nucleic acid and/orinserted at the target genomic locus can encode a polypeptide, canencode an miRNA, or it can comprise any regulatory regions or non-codingregions of interest including, for example, a regulatory sequence, apromoter sequence, an enhancer sequence, a transcriptionalrepressor-binding sequence, or a deletion of a non-protein-codingsequence, but does not comprise a deletion of a protein-coding sequence.In addition, the polynucleotide of interest within the insert nucleicacid and/or inserted at the target genomic locus can encode a proteinexpressed in the nervous system, the skeletal system, the digestivesystem, the circulatory system, the muscular system, the respiratorysystem, the cardiovascular system, the lymphatic system, the endocrinesystem, the urinary system, the reproductive system, or a combinationthereof. In one embodiment, the polynucleotide of interest within theinsert nucleic acid and/or inserted at the target genomic locus encodesa protein expressed in a bone marrow or a bone marrow-derived cell. Inone embodiment, the polynucleotide of interest within the insert nucleicadd and/or integrated at the target locus encodes a protein expressed ina spleen cell. In still further embodiments, the polynucleotide ofinterest within the insert nucleic acid and/or inserted at the targetlocus encodes a protein expressed in a B cell, encodes a proteinexpressed in an immature B cell or encodes a protein expressed in amature B cell.

The polynucleotide of interest within the insert polynucleotide cancomprise a portion of an ApoE locus, an IL-2-Rg locus, a Rag1 locus, aRag2 locus and/or a Rag2/Rag1 locus. Such portions of these given lociare discussed elsewhere herein, as are the various homologous andorthologous regions from any organism of interest that can be employed.

In one embodiment, polynucleotide of interest within the insert nucleicacid and/or inserted at the target locus comprises a genomic nucleicacid sequence that encodes an immunoglobulin heavy chain variable regionamino acid sequence. The phrase “heavy chain,” or “immunoglobulin heavychain” are described elsewhere herein.

In one embodiment, the polynucleotide of interest within the insertnucleic acid and/or integrated at the target locus comprises a genomicnucleic acid sequence that encodes a human immunoglobulin heavy chainvariable region amino acid sequence.

In one embodiment, the genomic nucleic acid sequence comprises one ormore unrearranged human immunoglobulin heavy chain gene segments, one ormore unrearranged human immunoglobulin heavy chain D gene segments, andone or more unrearranged human immunoglobulin heavy chain Ju genesegments, which are operably linked to a mammalian heavy chain constantregion nucleic acid sequence. In one embodiment, the genomic nucleicacid sequence comprises a rearranged human immunoglobulin heavy chainvariable region nucleic acid sequence operably linked to a mammalianheavy chain constant region nucleic acid sequence. In one embodiment,the genomic nucleic acid sequence comprises one or more unrearrangedhuman immunoglobulin V_(κ) or V_(λ) gene segments and one or moreunrearranged human immunoglobulin J_(κ) or J_(λ) gene segments, whichare operably linked to a mammalian immunoglobulin λ or κ light chainlight chain constant region nucleic acid sequence. In one embodiment,the genomic nucleic acid sequence comprises a rearranged humanimmunoglobulin λ or κ light chain variable region nucleic acid sequenceoperably linked to a mammalian immunoglobulin λ or κ light chain lightchain constant region nucleic acid sequence. In one embodiment, theheavy chain constant region nucleic acid sequence comprises a ratconstant region nucleic acid sequence, a human constant region nucleicacid sequence, or a combination thereof. In one embodiment, theimmunoglobulin λ or κ light chain constant region nucleic acid comprisesa rat constant region nucleic acid sequence, a human constant regionnucleic acid sequence, or a combination thereof.

In one embodiment, the immunoglobulin heavy chain constant regionnucleic acid sequence is selected from or comprises a CH1, a hinge, aCH2, a CH3, and/or a combination thereof. In one embodiment, the heavychain constant region nucleic acid sequence comprises aCH1-hinge-CH2-CH3.

In one embodiment, the polynucleotide of interest within the insertnucleic acid and/or integrated at the target locus comprises a genomicnucleic acid sequence that encodes an immunoglobulin light chainvariable region amino acid sequence. The phrase “light chain” includesan immunoglobulin light chain sequence from any organism, and isdescribed elsewhere herein.

In one embodiment, the polynucleotide of interest within the insertnucleic acid and/or integrated at the target genomic locus comprises agenomic nucleic acid sequence that encodes a human immunoglobulin lightchain variable region amino acid sequence.

one embodiment, the genomic nucleic acid sequence comprises one or moreunrearranged human immunoglobulin V_(κ) or V_(λ) gene segments and oneor more unrearranged human immunoglobulin J_(κ) or J_(λ) gene segments,which are operably linked to a rodent immunoglobulin λ or κ light chainlight chain constant region nucleic acid sequence. In one embodiment,the genomic nucleic acid sequence comprises a rearranged humanimmunoglobulin λ or κ light chain variable region nucleic acid sequenceoperably linked to a rodent immunoglobulin λ or κ light chain lightchain constant region nucleic acid sequence. In one embodiment, thelight chain constant region nucleic acid sequence comprises a ratconstant region nucleic acid sequence, a human constant region nucleicacid sequence, or a combination thereof. In one embodiment, theimmunoglobulin λ or κ light chain constant region nucleic acid comprisesa rat constant region nucleic acid sequence, a human constant regionnucleic acid sequence, or a combination thereof.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can encode an extracellular protein or aligand for a receptor. In specific embodiments, the encoded ligand is acytokine. Cytokines of interest includes a chemokine selected from orcomprising CCL, CXCL, CX3CL, and/or XCL. The cytokine can also comprisea tumor necrosis factor (TNF). In still other embodiments, the cytokineis an interleukin (IL). In one embodiment, the interleukin is selectedfrom or comprises IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IL-34, IL-35, and/or IL-36. In one embodiment, theinterleukin is IL-2. In specific embodiments, such polynucleotides ofinterest within the insert nucleic acid and/or integrated at the targetgenomic locus are from a human and, in more specific embodiments, cancomprise human genomic sequence.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target genomic locus can encode Apolipoprotein E(ApoE).

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can encode a cytoplasmic protein or amembrane protein. In one embodiment, the membrane protein is a receptor,such as, a cytokine receptor, an interleukin receptor, an interleukin 2receptor-alpha, an interleukin-2 receptor beta, an interleukin-2receptor gamma or receptor tyrosine kinase. In other instances, thepolynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can comprise an orthologous or homologousregion of the target locus.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can comprise a polynucleotide encoding atleast a region of a T cell receptor, including the T cell receptoralpha. In specific methods each of the insert nucleic acids comprise agenomic region of the T cell receptor locus (i.e. the T cell receptoralpha locus) such that upon completion of the serial integration, aportion or the entirety of the genomic T cell receptor locus has beenintegrated at the target locus. Such insert nucleic acids can compriseat least one or more of a variable segment or a joining segment of a Tcell receptor locus (i.e. of the T cell receptor alpha locus). In stillfurther embodiments, the polynucleotide of interest encoding the regionof the T cell receptor can be from, for example, a mammal, a non-humanmammal, rodent, mouse, rat, a human, a monkey, an agricultural mammal ora domestic mammal polynucleotide encoding a mutant protein.

In other embodiments, the polynucleotide of interest integrated at thetarget locus encodes a nuclear protein. In one embodiment, the nuclearprotein is a nuclear receptor. In specific embodiments, suchpolynucleotides of interest within the insert nucleic acid and/orintegrated at the target locus are from a human and, in more specificembodiments, can comprise human genomic sequence.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target genomic locus can comprise a geneticmodification in a coding sequence. Such genetic modifications include,but are not limited to, a deletion mutation of a coding sequence or thefusion of two coding sequences.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can comprise a polynucleotide encoding amutant protein, including, for example, a human mutant protein. In oneembodiment, the mutant protein is characterized by an altered bindingcharacteristic, altered localization, altered expression, and/or alteredexpression pattern. In one embodiment, the polynucleotide of interestwithin the insert nucleic acid and/or integrated at the target locuscomprises at least one disease allele, including for example, an alleleof a neurological disease, an allele of a cardiovascular disease, anallele of a kidney disease, an allele of a muscle disease, an allele ofa blood disease, an allele of a cancer-causing gene, or an allele of animmune system disease. In such instances, the disease allele can be adominant allele or the disease allele is a recessive allele. Moreover,the disease allele can comprises a single nucleotide polymorphism (SNP)allele. The polynucleotide of interest encoding the mutant protein canbe from any organism, including, but not limited to, a mammal, anon-human mammal, rodent, mouse, rat, a human, a monkey, an agriculturalmammal or a domestic mammal polynucleotide encoding a mutant protein.

In one embodiment, the genetic modification produces a mutant form of aprotein with an altered binding characteristic, altered localization,altered expression, and/or altered expression pattern.

In one embodiment, the genetic modification produces a deletion,addition, replacement or a combination thereof of a region of the ratApoE locus, wherein the genetic modification at the ApoE locus resultsin a decrease in ApoE activity. In one embodiment, an ApoE knockout isgenerated.

In one embodiment, the genetic modification produces a deletion,addition, replacement or a combination thereof of a region of the ratRag1 locus, wherein the genetic modification at the Rag1 locus resultsin a decrease in Rag1 activity. In one embodiment, a Rag1 knockout isgenerated. In one embodiment, the genetic modification produces adeletion, addition, replacement or a combination thereof of a region ofthe rat Rag2 locus, wherein the genetic modification at the Rag2 locusresults in a decrease in Rag2 activity. In one embodiment, a Rag2knockout is generated. In one embodiment, the genetic modificationproduces a deletion, addition, replacement or a combination thereof of aregion of the rat Rag1/Rag2 locus, wherein the genetic modification atthe Rag1/Rag2 locus results in a decrease in Rag1 activity and adecrease in Rag2 activity. In one embodiment, a Rag1/Rag2 knockout isgenerated.

In one embodiment, the genetic modification produces a deletion,addition, replacement or a combination thereof of a region of the ratinterleukin-2 receptor gamma locus, wherein the genetic modification atthe interleukin-2 receptor gamma locus results in a decrease ininterleukin-2 receptor gamma. In one embodiment, an interleukin-2receptor gamma knockout is generated.

As discussed elsewhere herein, further embodiments provided hereincomprises one or more of the rat ApoE locus, the rat interleukin-2receptor gamma locus, the Rag2 locus, the Rag1 locus and/or theRag2/Rag1 locus is modified through the replacement of a portion of therat ApoE locus, the interleukin-2 receptor gamma locus, the Rag2 locus,the Rag1 locus and/or Rag2/Rag1 locus with the corresponding orthologousportion of an ApoE locus, an interleukin-2 receptor gamma locus, a Rag2locus, a Rag1 locus and/or a Rag2/Rag1 locus from another organism.

In one embodiment, multiple genetic modifications are generated. In oneembodiment, a genetic modification produces a deletion, addition,replacement or a combination thereof of a region of the ratinterleukin-2 receptor gamma locus, wherein the genetic modification atthe interleukin-2 receptor gamma locus results in a decrease ininterleukin-2 receptor gamma and a second genetic modification producesa deletion, addition, replacement or a combination thereof of a regionof the rat Rag2 locus, wherein the genetic modification at the Rag2locus results in a decrease in Rag2 activity. In one embodiment, aninterleukin-2 receptor gamma/Rag2 knockout is generated. Such a rat hasa SCID phenotype.

In one embodiment, the mammalian nucleic acid comprises a genomic locusthat encodes a protein expressed in the nervous system, the skeletalsystem, the digestive system, the circulatory system, the muscularsystem, the respiratory system, the cardiovascular system, the lymphaticsystem, the endocrine system, the urinary system, the reproductivesystem, or a combination thereof. In one embodiment, the mammaliannucleic acid comprises a genomic locus that encodes a protein expressedin a bone marrow or a bone marrow-derived cell. In one embodiment, thenucleic acid comprises a genomic locus that encodes a protein expressedin a spleen cell. In one embodiment, the genomic locus comprises a mousegenomic DNA sequence, a rat genomic DNA sequence a human genomic DNAsequence, or a combination thereof. In one embodiment, the genomic locuscomprises, in any order, rat and human genomic DNA sequences. In oneembodiment, the genomic locus comprises, in any order, mouse and humangenomic DNA sequences. In one embodiment, the genomic locus comprises,in any order, mouse and rat genomic DNA sequences. In one embodiment,the genomic locus comprises, in any order, rat, mouse, and human genomicDNA sequences.

In one embodiment, the insert nucleic acid comprises a geneticmodification in a coding sequence of a gene. In one embodiment, thegenetic modification comprises a deletion mutation in the codingsequence. In one embodiment, the genetic modification comprises a fusionof two endogenous coding sequences.

In one embodiment, the genetic modification comprises a deletion of anon-protein-coding sequence, but does not comprise a deletion of aprotein-coding sequence. In one embodiment, the deletion of thenon-protein-coding sequence comprises a deletion of a regulatoryelement. In one embodiment, the genetic modification comprises anaddition of a promoter. In one embodiment, the genetic modificationcomprises a replacement of a promoter or regulatory element. In oneembodiment, the regulatory element is an enhancer. In one embodiment,the regulatory element is a transcriptional repressor-binding element.

In one embodiment, the genetic modification comprises placement of ahuman nucleic acid sequence encoding a mutant human protein. In oneembodiment, the genetic modification comprises at least one humandisease allele of a human gene. In one embodiment, the human disease isa neurological disease. In one embodiment, the human disease is acardiovascular disease. In one embodiment, the human disease is a kidneydisease. In one embodiment, the human disease is a muscle disease. Inone embodiment, the human disease is a blood disease. In one embodiment,the human disease is a cancer. In one embodiment, the human disease isan immune system disease. In one embodiment, the human disease allele isa dominant allele. In one embodiment, the human disease allele is arecessive allele. In one embodiment, the human disease allele comprisesa single nucleotide polymorphism (SNP) allele.

The polynucleotide of interest within the insert nucleic acid and/orintegrated at the target locus can also comprise a regulatory sequence,including for example, a promoter sequence, an enhancer sequence, or atranscriptional repressor-binding sequence. In specific embodiments, thepolynucleotide of interest within the insert nucleic acid and/orintegrated at the target genomic locus comprises a polynucleotide havinga deletion of a non-protein-coding sequence, but does not comprise adeletion of a protein-coding sequence. In one embodiment, the deletionof the non-protein-coding sequence comprises a deletion of a regulatorysequence. In another embodiment, the deletion of the regulatory elementcomprises a deletion of a promoter sequence. In one embodiment, thedeletion of the regulatory element comprises a deletion of an enhancersequence. Such a polynucleotide of interest can be from any organism,including, but not limited to, a mammal, a non-human mammal, rodent,mouse, rat, a human, a monkey, an agricultural mammal or a domesticmammal polynucleotide encoding a mutant protein.

5. Methods of Introducing Sequences and Generation of Transgenic Animals

As outlined above, methods and compositions are provided herein to allowfor the targeted integration of one or more polynucleotides of interestinto a target locus. Such systems employ a variety of components and forease of reference, herein the term “targeted integration system”generically comprises all the components required for an integrationevent (i.e. in non-limiting examples, the various nuclease agents,recognition sites, insert DNA polynucleotides, targeting vectors, targetgenomic locus, and/or polynucleotides of interest).

The methods provided herein comprise introducing into a cell one or morepolynucleotides or polypeptide constructs comprising the variouscomponents of the targeted genomic integration system. “Introducing”means presenting to the cell the sequence (polypeptide orpolynucleotide) in such a manner that the sequence gains access to theinterior of the cell. The methods provided herein do not depend on aparticular method for introducing any component of the targeted genomicintegration system into the cell, only that the polynucleotide gainsaccess to the interior of a least one cell. Methods for introducingpolynucleotides into various cell types are known in the art andinclude, but are not limited to, stable transfection methods, transienttransfection methods, and virus-mediated methods.

in some embodiments, the cells employed in the methods and compositionshave a DNA construct stably incorporated into their genome. “Stablyincorporated” or “stably introduced” means the introduction of apolynucleotide into the cell such that the nucleotide sequenceintegrates into the genome of the cell and is capable of being inheritedby progeny thereof. Any protocol may be used for the stableincorporation of the DNA constructs or the various components of thetargeted genomic integration system.

Transfection protocols as well as protocols for introducing polypeptidesor polynucleotide sequences into cells may vary. Non-limitingtransfection methods include chemical-based transfection methods includethe use of liposomes; nanoparticles; calcium phosphate (Graham et al.(1973). Virology 52 (2): 456-67, Bacchetti et al. (1977) Proc Natl AcadSci USA 74 (4): 1590-4 and, Kriegler, M (1991). Transfer and Expression:A Laboratory Manual. New York: W. H. Freeman and Company. pp. 96-97);dendrimers; or cationic polymers such as DEAE-dextran orpolyethylenimine. Non chemical methods include electroporation;Sono-poration; and optical transfection Particle-based transfectioninclude the use of a gene gun, magnet assisted transfection (Bertram, J.(2006) Current Pharmaceutical Biotechnology 7, 277-28). Viral methodscan also be used for transfection.

In one embodiment, the introducing one or more of the polynucleotidesinto a cell is mediated by electroporation, by intracytoplasmicinjection, by a viral infection, by an adenovirus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection or ismediated via Nucleofection™.

In one embodiment, introduction one or more of the polynucleotides intoa cell further comprises: introducing an expression construct comprisinga nucleic acid sequence of interest operably linked to a promoter. Inone embodiment, the promoter is a constitutively-active promoter. In oneembodiment, the promoter is an inducible promoter. In one embodiment,the promoter is active in the rat embryonic stem cell.

In one embodiment, the expression construct is introduced together withthe LTVEC. In one embodiment, the expression construct is introducedseparately from the LTVEC over a period of time.

In one embodiment, the introduction of the one or more polynucleotidesinto the cell can be performed multiple times over a period of time. Inone embodiment, the introduction of the one or more polynucleotides intothe cell are performed at least two times over a period of time, atleast three times over a period of time, at least four times over aperiod of time, at least five times over a period of time, at least sixtimes over a period of time, at least seven times over a period of time,at least eight times over a period of time, at least nine times over aperiod of times, at least ten times over a period of time, at leasteleven times, at least twelve times over a period of time, at leastthirteen times over a period of time, at least fourteen times over aperiod of time, at least fifteen times over a period of time, at leastsixteen times over a period of time, at least seventeen times over aperiod of time, at least eighteen times over a period of time, at leastnineteen times over a period of time, or at least twenty times over aperiod of time.

In one embodiment, the nuclease agent is introduced into the c ellsimultaneously with the targeting vector or the large targeting vector(LTVEC). Alternatively, the nuclease agent is introduced separately fromthe targeting vector or the LTVEC over a period of time. In oneembodiment, the nuclease agent is introduced prior to the introductionof the targeting vector or the LTVEC, while in other embodiments, thenuclease agent is introduced following introduction of the targetingvector or the LTVEC.

In one embodiment, screening step comprises a quantitative assay forassessing modification of allele (MOA) of a parental chromosome. In oneembodiment, the quantitative assay is carried out via a quantitativePCR. In one embodiment, the quantitative PCR is a real-time PCR (qPCR).In one embodiment, the real-time PCR comprises a first primer set thatrecognizes the target locus and a second primer set that recognizes anon-targeted reference locus. In one embodiment, the primer setcomprises a fluorescent probe that recognizes the amplified sequence. Inone embodiment, the quantitative assay is carried out viafluorescence-mediated in situ hybridization (FISH). In one embodiment,the quantitative assay is carried out via comparative genomichybridization. In one embodiment, the quantitative assay is carried outvia isothermic DNA amplification. In one embodiment, the quantitativeassay is carried out via isothermic DNA amplification. In oneembodiment, the quantitative assay is carried out via quantitativehybridization to an immobilized probe(s). In one embodiment, thequantitative assay is carried out via Invader Probes®. In oneembodiment, the quantitative assay is carried out via MMP Assays®. Inone embodiment, the quantitative assay is carried out via TaqMan®Molecular Beacon. In one embodiment, the quantitative assay is carriedout via Eclipse™ probe technology. (See, for example, US2005/0144655,which is incorporated by reference herein in its entirety).

Further provided is a method for making a humanized rat, comprising: (a)modifying a genome of a pluripotent rat cell with a targeting vectorcomprising an insert nucleic acid that comprises a human nucleic acidsequence to form a donor cell; (b) introducing the donor cell into ahost rat embryo; and (c) gestating the host rat embryo in a surrogatemother; wherein the surrogate mother produces a rat progeny thatcomprises the human nucleic acid sequence. In one embodiment, the donorcell is introduced into a host rat embryo that is at the blastocyststage or at a pre-morula stage (i.e., a 4 cell stage or an 8 cellstage). Moreover, step (a) can also be performed with a large targetingvector (LTVEC) and/or a human nucleic acid sequence at least 5 Kb inlength. In still further embodiments, the genetic modification iscapable of being transmitted through the germline.

Genetically modified rats can be generated employing the various methodsdisclosed herein. Such methods comprise (1) integrating one or morepolynucleotide of interest at the target locus of a pluripotent rat cellto generate a genetically modified pluripotent rat cell comprising theinsert nucleic acid in the targeted genomic locus employing the methodsdisclosed herein; (2) selecting the genetically modified pluripotent ratcell having the one or more polynucleotides of interest at the targetgenomic locus; (3) introducing the genetically modified pluripotent ratcell into a rat host embryo; and (4) implanting the host rat embryocomprising the genetically modified pluripotent rat cell into asurrogate mother. A progeny from the genetically modified pluripotentrat cell is generated. In one embodiment, the donor cell is introducedinto a rat host embryo at the blastocyst stage or at the pre-morulastage (i.e., the 4 cell stage or the 8 cell stage). Progeny that arecapable of transmitting the genetic modification though the germline aregenerated. The pluripotent rat cell can be a rat ES cell as discussedelsewhere herein.

Nuclear transfer techniques can also be used to generate the geneticallymodified rats. Briefly, methods for nuclear transfer include the stepsof: (1) enucleating oocyte; (2) isolating a donor cell or nucleus to becombined with the enucleated oocyte; (3) inserting the cell or nucleusinto the enucleated oocyte to form a reconstituted cell; (4) implantingthe reconstituted cell into the womb of an animal to form an embryo; and(5) allowing the embryo to develop. In such methods oocytes aregenerally retrieved from deceased animals, although they may be isolatedalso from either oviducts and/or ovaries of live animals. Oocytes can bematured in a variety of medium known to those of ordinary skill in theart prior to enucleation. Enucleation of the oocyte can be performed ina number of manners well known to those of ordinary skill in the art.Insertion of the donor cell or nucleus into the enucleated oocyte toform a reconstituted cell is usually by microinjection of a donor cellunder the zona pellucida prior to fusion. Fusion may be induced byapplication of a DC electrical pulse across the contact/fusion plane(electrofusion), by exposure of the cells to fusion-promoting chemicals,such as polyethylene glycol, or by way of an inactivated virus, such asthe Sendai virus. A reconstituted cell is typically activated byelectrical and/or non-electrical means before, during, and/or afterfusion of the nuclear donor and recipient oocyte. Activation methodsinclude electric pulses, chemically induced shock, penetration by sperm,increasing levels of divalent cations in the oocyte, and reducingphosphorylation of cellular proteins (as by way of kinase inhibitors) inthe oocyte. The activated reconstituted cells, or embryos, are typicallycultured in medium well known to those of ordinary skill in the art andthen transferred to the womb of an animal. See, for example,US20080092249, WO/1999/005266A2, US20040177390, WO/2008/017234A1, andU.S. Pat. No. 7,612,250, each of which is herein incorporated byreference.

In one aspect, a method for making a genetically modified rat isprovided, comprising modifying a genomic locus of interest in apluripotent rat cell employing endonuclease-mediated gene targeting tointroduce a modification at a rat genomic locus of interest to form amodified pluripotent rat cell, maintaining the modified pluripotent ratcell under conditions sufficient to maintain pluripotency, employing themodified pluripotent rat cell as a donor cell in a rat host embryo, andgestating the host embryo comprising the modified pluripotent rat cellin a surrogate mother, wherein the host embryo is gestated by thesurrogate mother and a genetically modified rat progeny is born.

In one embodiment, the target sequence is located in an intron. In oneembodiment, the target sequence is located in an exon. In oneembodiment, the target sequence is located in a promoter. In oneembodiment, the target sequence is located in a promoter regulatoryregion. In one embodiment, the target sequence is located in an enhancerregion.

In one embodiment, introducing step is performed multiple times over aperiod of time using a plurality of endonucleases that recognizedistinct target sequences. In one embodiment, step is performed at leasttwo times over a period of time using a plurality of endonucleases thatrecognize distinct target sequences, at least three times over a periodof time using a plurality of endonucleases that recognize distincttarget sequences, at least four times over a period of time using aplurality of endonucleases that recognize distinct target sequences, atleast five times over a period of time using a plurality ofendonucleases that recognize distinct target sequences, at least sixtimes over a period of time using a plurality of endonucleases thatrecognize distinct target sequences, at least seven times over a periodof time using a plurality of endonucleases that recognize distincttarget sequences, at least eight times over a period of time using aplurality of endonucleases that recognize distinct target sequences, atleast nine times over a period of time using a plurality ofendonucleases that recognize distinct target sequences, at least tentimes over a period of time using a plurality of endonucleases thatrecognize distinct target sequences, at least eleven times over a periodof time using a plurality of endonucleases that recognize distincttarget sequences, at least twelve times over a period of time using aplurality of endonucleases that recognize distinct target sequences, atleast thirteen times over a period of time using a plurality ofendonucleases that recognize distinct target sequences, at leastfourteen times over a period of time using a plurality of endonucleasesthat recognize distinct target sequences, at least fifteen times over aperiod of time using a plurality of endonucleases that recognizedistinct target sequences, at least sixteen times over a period of timeusing a plurality of endonucleases that recognize distinct targetsequences, at least seventeen times over a period of time using aplurality of endonucleases that recognize distinct target sequences, atleast eighteen times over a period of time using a plurality ofendonucleases that recognize distinct target sequences, at leastnineteen times over a period of time using a plurality of endonucleasesthat recognize distinct target sequences, or at least twenty times overa period of time using a plurality of endonucleases that recognizedistinct target sequences.

In one embodiment, introducing step is mediated by electroporation, byintracytoplasmic injection, by an adenovirus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection or ismediated via Nucleofection™.

In one embodiment, the method further comprises introducing an exogenousnucleic acid into the genetically modified pluripotent rat cell. In oneembodiment, the exogenous nucleic acid is a transgene. In oneembodiment, the exogenous nucleic acid is introduced into an endogenouslocus. In one embodiment, the exogenous nucleic acid is introducedectopically (e.g., at a locus different from its endogenous locus).

In one aspect, a method for making a genetically modified rat isprovided, comprising modifying a genomic locus of interest in apluripotent rat cell employing RNA-guided genome engineering tointroduce a modification at a rat genomic locus of interest to form amodified pluripotent rat cell, maintaining the modified pluripotent ratcell under conditions sufficient to maintain pluripotency, employing themodified pluripotent rat cell as a donor cell in a rat host embryo, andgestating the host embryo comprising the modified pluripotent rat cellin a surrogate mother, wherein the host embryo is gestated by thesurrogate mother and a genetically modified rat progeny is born.

In one embodiment, the method has a targeting rate ranging from about 2%to about 80%.

In one embodiment, the method comprises co-introducing a plurality ofthe second expression construct comprising distinct genomic targetsequences for multiplex editing of distinct genomic loci. In onembodiment, the method comprises introducing a plurality of the secondexpression construct comprising distinct genomic target sequences formultiplex editing of distinct genomic loci over a period of time.

In one embodiment, introducing step is performed multiple times over aperiod of time. In one embodiment, introducing step (b) is performed atleast two times over a period of time, at least three times over aperiod of time, at least four times over a period of time, at least fivetimes over a period of time, at least six times over a period of time,at least seven times over a period of time, at least eight times over aperiod of time, at least nine times over a period of time, at least tentimes over a period of time, at least eleven times over a period oftime, at least twelve times over a period of time, at least thirteentimes over a period of time, at least fourteen times over a period oftime, at least fifteen times over a period of time, at least sixteentimes over a period of time, at least seventeen times over a period oftime, at least eighteen times over a period of time, at least nineteentimes over a period of time, at least twenty times over a period oftime.

In one embodiment, the first expression construct and the secondexpression construct are expressed from a same plasmid.

In one embodiment, introducing step is mediated by electroporation, byintracytoplasmic injection, by an adenovirus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection or ismediated via Nucleofection™.

In one embodiment, the method further comprises introducing an exogenousnucleic acid into the pluripotent rat cell comprising the mutant allele.

In one embodiment, the exogenous nucleic acid is a transgene. In oneembodiment, the exogenous nucleic acid is introduced into an endogenouslocus. In one embodiment, the exogenous nucleic acid is placedectopically (e.g., at a locus different from its endogenous locus).

In one embodiment, the method further comprises introducing an exogenousnucleic acid into the genetically modified pluripotent rat cell. In oneembodiment, the exogenous nucleic acid is a transgene. In oneembodiment, the exogenous nucleic acid is introduced into an endogenouslocus. In one embodiment, the exogenous nucleic acid is introducedectopically (e.g., at a locus different from its endogenous locus).

In one aspect, a method for making a humanized rat is provided,comprising modifying a genome of a pluripotent rat cell with an LTVECcomprising an insert that comprises a human sequence of at least 5 kb,and employing the pluripotent rat cell as a donor cell, introducing thedonor cell into a host embryo, and gestating the host embryo in asurrogate mother, wherein the surrogate mother births a rat progeny thatcomprises the humanization.

Other methods for making a genetically modified rat comprising in itsgermline one or more genetic modifications as described herein isprovided, comprising: (a) modifying a targeted rat locus contained in aprokaryotic cell employing the various methods described herein; (b)selecting a modified prokaryotic cell comprising the geneticmodification at the targeted rat locus; (c) isolating the geneticallymodified targeting vector from the genome of the modified prokaryoticcell; (d) introducing the genetically modified targeting vector into apluripotent rat cell to generate a genetically modified pluripotent cellcomprising the insert nucleic acid at the targeted genomic locus; (e)selecting the genetically modified rat pluripotent cell; (f) introducingthe genetically modified pluripotent rat cell into a host rat embryo ata pre-morula stage; and (g) implanting the host rat embryo comprisingthe genetically modified pluripotent rat cell into a surrogate mother togenerate an F0 generation derived from the genetically modifiedpluripotent rat cell. In such methods the targeting vector can comprisea large targeting vector. The pluripotent rat cell can be a rat ES cell.In further methods, the isolating step (c) further comprises (c1)linearizing the genetically modified targeting vector (i.e., thegenetically modified LTVEC). In still further embodiments, theintroducing step (d) further comprises (d1) introducing a nuclease agentas described herein into the pluripotent rat cell. In one embodiment,selecting steps (b) and/or (e) are carried out by applying a selectableagent as described herein to the prokaryotic cell or the pluripotent ratcell. In one embodiment, selecting steps (b) and/or (e) are carried outvia a modification of allele (MOA) assay as described herein.

Further methods for modifying a target genomic locus of a mammalian cellvia bacterial homologous recombination (BHR) in a prokaryotic cell areprovided and comprise: (a) providing a prokaryotic cell comprising atarget locus comprising a rat nucleic acid, (b) introducing into theprokaryotic cell a targeting vector comprising an insert nucleic acidflanked with a 5′ rat homology arm and a 3′ rat homology arm, whereinthe insert nucleic acid comprises a mammalian region (including, forexample, a. DNA insert from a human), and (c) selecting a targetedprokaryotic cell comprising the insert nucleic acid at the target ratlocus, wherein the prokaryotic cell is capable of expressing arecombinase that mediates the BHR. Step (a1) can comprise providing aprokaryotic cell comprising a target locus comprising a rat nucleic acidcomprising a first polynucleotide comprising a first recognition sitefor a first nuclease agent, and step (hi) can further compriseexpressing in the prokaryotic cell a nuclease agent that makes a nick ordouble-strand break at or near the first recognition site. Steps (a)-(c)can be serially repeated as disclosed herein to allow the introductionof multiple insert nucleic acids at the targeted rat locus in theprokaryotic cell. Once the targeted genomic locus is “built” with theprokaryotic cell, a targeting vector comprising the modified target ratlocus can be isolated from the prokaryotic cell and introduced into atarget genomic locus within a pluripotent rat cell. Pluripotent ratcells (i.e., rat ES cells) comprising the modified genomic locus canthen be made into genetically modified rats.

In some embodiments, various genetic modifications of the target genomicloci described herein can be carried out by a series of homologousrecombination reactions (BHR) in bacterial cells using an LTVEC derivedfrom Bacterial Artificial Chromosome (BAC) DNA using VELOCIGENE® geneticengineering technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuela, D. M. et al. (2003), High-throughput engineering of themouse genome coupled with high-resolution expression analysis, NatureBiotechnology 21(6): 652-659, which is incorporated herein by referencein their entireties).

In some embodiments, targeted rat ES cells comprising various geneticmodifications as described herein are used as insert ES cells andintroduced into a pre-morula stage embryo from a corresponding organism,e.g., an 8-cell stage mouse embryo, via the VELOCIMOUSE® method (see,e.g., U.S. Pat. Nos. 7,576,259, 7,659,442, 7,294,754, and US2008-0078000 A1, all of which are incorporated by reference herein intheir entireties). The rat embryo comprising the genetically modifiedrat ES cells is incubated until the blastocyst stage and then implantedinto a surrogate mother to produce an F0. Rats bearing the geneticallymodified genomic locus can be identified via modification of allele(MOA) assay as described herein. The resulting F0 generation rat derivedfrom the genetically modified ES rat cells is crossed to a wild-type ratto obtain F1 generation offspring. Following genotyping with specificprimers and/or probes, F1 rats that are heterozygous for the geneticallymodified genomic locus are crossed to each other to produce rats thatare homozygous for the genetically modified genomic locus.Alternatively, an F0 female rat and an F0 male rat each having thegenetic modification can be crossed to obtain an F1 rat homozygous forthe genetic modification.

In one aspect, a genetically modified rat genome is provided, comprisinga targeted modification of an endogenous rat nucleic acid sequence witha homologous or orthologous non-rat nucleic acid sequence.

In one embodiment, the homologous or orthologous non-rat nucleic acidsequence is of a length from about 5 kb to about 200 kb. In oneembodiment, the homologous or orthologous non-rat nucleic acid sequenceranges from about 5 kb to about 10 kb. In one embodiment, the homologousor orthologous non-rat nucleic acid sequence ranges from about 10 kb toabout 20 kb. In one embodiment, the homologous or orthologous non-ratnucleic acid sequence ranges from about 20 kb to about 30 kb. In oneembodiment, the homologous or orthologous non-rat nucleic acid sequenceranges from about 30 kb to about 40 kb. In one embodiment, thehomologous or orthologous non-rat nucleic acid sequence ranges fromabout 40 kb to about 50 kb. In one embodiment, the homologous ororthologous non-rat nucleic acid sequence ranges from about 50 kb toabout 60 kb. In one embodiment, the homologous or orthologous non-ratnucleic acid sequence ranges from about 60 kb to about 70 kb. In oneembodiment, the homologous or orthologous non-rat nucleic acid sequenceranges from about 70 kb to about 80 kb. In one embodiment, thehomologous or orthologous non-rat nucleic acid sequence ranges fromabout 80 kb to about 90 kb. In one embodiment, the homologous ororthologous non-rat nucleic acid sequence ranges from about 90 kb toabout 100 kb. In one embodiment, the homologous or orthologous non-ratnucleic acid sequence ranges from about 100 kb to about 110 kb. In oneembodiment, the homologous or orthologous non-rat nucleic acid sequenceranges from about 110 kb to about 120 kb. In one embodiment, thehomologous or orthologous non-rat nucleic acid sequence ranges fromabout 120 kb to about 130 kb. In one embodiment, the homologous ororthologous non-rat nucleic acid sequence ranges from about 140 kb toabout 150 kb. In one embodiment, the homologous or orthologous non-ratnucleic acid sequence ranges from about 150 kb to about 160 kb. In oneembodiment, the homologous or orthologous non-rat nucleic acid sequenceranges from about 160 kb to about 170 kb. In one embodiment, thehomologous or orthologous non-rat nucleic acid sequence ranges fromabout 170 kb to about 180 kb. In one embodiment, the homologous ororthologous non-rat nucleic acid sequence ranges from about 180 kb toabout 190 kb. In one embodiment, the homologous or orthologous non-ratnucleic acid sequence ranges from about 190 kb to about 200 kb. Variouspolynucleotides of interest that can be employed in the insert nucleicacid are described elsewhere herein.

6. Cells

The various methods and compositions described herein employ a genomiclocus targeting system in a cell. In one embodiment, the cell is apluripotent cell. In one embodiment, the pluripotent cell is a non-humanpluripotent cell. In one embodiment, the non-human pluripotent cell is amammalian pluripotent cell. In one embodiment, the pluripotent cell is ahuman induced pluripotent stem (iPS) cell.

In one embodiment, the pluripotent cell is a pluripotent rat cell. Inone embodiment, the pluripotent rat cell is a rat embryonic stem (ES)cell. In one embodiment, the pluripotent rat cell is an inducedpluripotent stem (iPS) cell or is a developmentally restrictedprogenitor cell. In other embodiments, the pluripotent rat cell is ableto sustain its pluripotency following at least one targeted geneticmodification of its genome and is able to transmit the targetedmodification to a germline of an F1 generation.

In one embodiment, the pluripotent cell is a non-human fertilized egg atthe single cell stage. In one embodiment, the non-human fertilized eggis a mammalian fertilized egg. In one embodiment, the mammalianfertilized egg is a rodent fertilized egg at the single cell stage. Inone embodiment, the mammalian fertilized egg is a rat or mousefertilized egg at the single cell stage.

The various cells employed in the method and compositions disclosedherein can also comprise prokaryotic cells, such as a bacterial cell,including E coli. In specific embodiments, the prokaryotic cell is arecombination-competent strain of E. coli. In one embodiment, theprokaryotic cell comprises a nucleic acid that encodes the recombinase,while in other instances, the prokaryotic cell does not comprise thenucleic acid that encodes the recombinase, and the nucleic acid encodingthe recombinase is introduced into the prokaryotic cell. In oneembodiment, the nucleic acid encoding the recombinase comprises a DNA oran mNA. In some embodiments, the nucleic acid encoding the recombinaseis pABG. In one embodiment, the recombinase is expressed under thecontrol of an inducible promoter. In one embodiment, expression of therecombinase is controlled by arabinose.

A. Rat Embryonic Stem (ES) Cells

As outlined in detail above, the various compositions and methodsprovided herein can employ embryonic stem (ES) cells from rat. In oneembodiment, the pluripotent rat cell is a rat ES cell. In oneembodiment, the rat ES cell is derived from a rat strain is a Wistarrat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344,1′6, and Dark Agouti or ACI. In one embodiment, the rat strain is a mixof two or more of a strain selected from the group consisting of Wistar,LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti. In oneembodiment, the rat ES cell is derived from an inbred strain. In oneembodiment, the rat ES cell is derived from a strain selected from a DAstrain and an ACI strain. In a specific embodiment, the rat ES cell isderived from an ACI strain. In one embodiment, the rat ES cell isderived from a rat blastocyst.

In other embodiments, the rat ES cell is characterized by expression ofat least one pluripotency marker. In specific embodiments, the rat EScell is characterized by expression of a pluripotency marker comprisingOct-4, Sox-2, alkaline phosphatase, or a combination thereof. In oneembodiment, the rat ES cell is a male (XY) rat ES cell or a female (XX)rat ES cell.

In one embodiment, following the one to 15 serial genetic modifications,the genetically modified rat ES cells upon exposure to differentiationmedium are capable of differentiation into a plurality of cell types.

In one embodiment, following the one to 15 serial genetic modifications,the genetically modified rat ES cells are capable of being maintained inan undifferentiated state in culture. In one embodiment, the geneticallymodified and cultured rat ES cells in the undifferentiated state, whenemployed as donor cells in a rat host embryo, populate the embryo andform a blastocyst comprising the one to fifteen genetic modifications.In one embodiment, the blastocyst, when implanted into a surrogatemother under conditions suitable for gestation, develops into an F0 ratprogeny that comprises the one to 15 genetic modifications.

In one aspect, an isolated rat ES cell is provided that is capable ofsustaining pluripotency following one or more genetic modifications invitro, and that is capable of transmitting a genetically modified genometo a germline of an F1 generation.

In one embodiment, the rat ES cell maintains its pluripotency to developinto a plurality of cell types following the one or more serial geneticmodifications in vitro (e.g., two, three, four, five, or six or moreserial genetic modifications). In one embodiment, the geneticmodification is mediated by an electroporation, by intracytoplasmicinjection, by a viral infection, by an adenovirus, by lentivirus, byretrovirus, by transfection, by lipid-mediated transfection, or byNucleofection™.

In one embodiment, the rat ES cell maintains its pluripotency to developinto a plurality of cell types following a single round ofelectroporation with an exogenous nucleic acid. In one embodiment, therat ES cell maintains its pluripotency to develop into a plurality ofcell types following a 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th), 7^(th),8^(th), 9^(th), 10^(th), 11^(th), 12^(th), 13^(th), 14^(th), or 15^(th)round of electroporation with an exogenous nucleic acid.

In other embodiments, the rat ES cells employed are those described inUS 2014-0235933, herein incorporated by reference in its entirety.

The pluripotent rat cell employed in the various methods andcompositions disclosed herein can be characterized by expression of atleast one pluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15,Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2,Utf1, and/or a combination thereof. In other instances, the pluripotentrat cell employed in the various methods and compositions disclosedherein is characterized by one or more of the following features: (a)lack of expression of one or more pluripotency markers comprising c-Myc,Ecat1, and/or Rexo1; (b) lack of expression of one or more mesodermalmarkers comprising Brachyury and/or Bmpr2; (c) lack of expression of oneor more endodermal markers comprising Gata6, Sox17, and/or Sox7; or (d)lack of expression of one or more neural markers comprising Nestinand/or Pax6. As used herein, “lack of expression” as it relates toexpression of a pluripotency marker means that the expression of thepluripotency marker is at or below the experimental background asdetermined for each individual experiment.

In one non-limiting embodiment, the rat ES cells provided herein haveone or more of any of the following properties:

(a) have germ-line competency, meaning when the rat ES cell is implantedinto a rat host embryo, the genome of the rat ES cell line istransmitted into an offspring;

(b) have germ-line competency following at least one targeted geneticmodification, meaning when the rat ES cell having the targeted geneticmodification is implanted into a rat host embryo, the targeted geneticmodification within the genome of the rat ES cell line is transmittedinto an offspring;

(c) have pluripotency in vitro;

(d) have totipotency in vitro;

(e) when cultured in vitro loosely adhere to a feeder cell layer;

when cultured in vitro form sphere-like colonies when plated on a feedercell layer in vitro;

(g) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer that is not genetically modified toexpress leukemia inhibitory factor (LIF), wherein the culture mediacomprises a sufficient concentration of LIF;

(h) maintain pluripotency when cultured in vitro under conditionscomprising a feeder cell layer, wherein the culture media comprisesmouse LIF or an active variant or fragment thereof;

(i) comprise a molecular signature that is characterized by

-   -   i) the expression of one or more of rat ES cell-specific genes        comprising Adheres Junctions Associate Protein (Ajap1), Claudin        5 (Cldn5), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9),        Calcium/calmodulin-dependent protein kinase IV (Camk4),        ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein        beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1        (Igfbpl1), Interleukin 36 beta(Il1f8), Interleukin 28 receptor,        alpha (Il28ra), left-right determination factor 1 (Lefty1),        Leukemia inhibitory factor receptor alpha (Lifr),        Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin        receptor (Ntm), Protein tyrosine phosphatase non-receptor type        18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III        and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid        transcription factor 2) (Foxe1), Hairy/enhancer-of-split related        with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription        factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW        motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1),        Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),        miR-632, or a combination thereof;    -   ii) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more        of the rat ES cell-specific genes comprising Adheres Junctions        Associate Protein (Ajap1), Claudin 5 (Cldn5), Cdc42 guanine        nucleotide exchange factor 9 (Arhgef9),        Calcium/calmodulin-dependent protein kinase IV (Camk4),        ephrin-A1 (Efna1), EPH receptor A4 (Epha4), gap junction protein        beta 5 (Gjb5), Insulin-like growth factor binding protein-like 1        (Igfbpl1), Interleukin 36 beta(Il1f8), Interleukin 28 receptor,        alpha (Il28ra), left-right determination factor 1 (Lefty1),        Leukemia inhibitory factor receptor alpha (Lifr),        Lysophosphatidic acid receptor 2 (Lpar2), Neuronal pentraxin        receptor (Ntm), Protein tyrosine phosphatase non-receptor type        18 (Ptpn18), Caudal type homeobox 2 (Cdx2), Fibronectin type III        and ankyrin repeat domains 1 (Fank1), Forkhead box E1 (thyroid        transcription factor 2) (Foxe1), Hairy/enhancer-of-split related        with YRPW motif 2 (Hey2), Forkhead box E1 (thyroid transcription        factor 2) (Foxe1), Hairy/enhancer-of-split related with YRPW        motif 2 (Hey2), Lymphoid enhancer-binding factor 1 (Lef1),        Sal-like 3 (Drosophila) (Sall3), SATB homeobox 1 (Satb1),        miR-632, or a combination thereof;    -   iii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 9        when compared to a F1H4 mouse ES cell;    -   iv) at least a 20-fold increase in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25 or more of the rat ES cell-specific genes as        set forth in Table 9 when compared to a F1H4 mouse ES cell;    -   v) the expression of one or more of rat ES cell-specific genes        as set forth in Table 10;    -   vi) the expression of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35,        40, 45, 50 or more of the rat ES cell-specific genes as set        forth in Table 10;    -   vii) at least a 20-fold increase in the expression of one or        more of the rat ES cell-specific genes as set forth in Table 10        when compared to a F1H4 mouse ES cell;    -   viii) at least a 20-fold increase in the expression of at least        2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,        20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 10 when compared to a        F1H4 mouse ES cell;    -   ix) at least a 20-fold decrease in the expression of one or more        of the rat ES cell-specific genes as set forth in Table 8 when        compared to a F1H4 mouse ES cell;    -   x) at least a 20-fold decrease in the expression of at least 2,        3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more of the rat ES        cell-specific genes as set forth in Table 8 when compared to a        F1H4 mouse ES cell;    -   xi) any combination of expression of the rat ES cell-specific        genes of parts (i)-(x);    -   xii) a relative expression level of pluripotency markers as        shown in Table 11 for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17 or 18 of the listed pluripotency markers.        See, pluripotency ranking column of Table 11 for relative        expression levels;    -   xiii) a relative expression level of the mesodermal markers as        shown in Table 11 for at least 2, 3, or 4 of the listed        mesodermal markers. See, mesodermal ranking column in Table 11        for relative expression levels;    -   xiv) a relative expression level of endodermal markers as shown        in Table 11 for at least 2, 3, 4, 5, or 6 of the listed        endodermal markers. See, endodermal ranking column in Table 11        for relative expression levels;    -   xv) a relative expression level of neural markers as shown in        Table 11 for at least 2 and 3 of the listed neural markers. See,        neural ranking column in Table 11 for relative expression        levels;    -   xvi) a relative expression level of trophectoderm markers as        shown in Table 11 for the listed trophectoderm markers. See,        trophectoderm ranking column in Table 11 for relative expression        levels;    -   xvii) any relative expression level of one or more (2, 3, 4, 5,        6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,        23, 24, 25, 26, 27, 28, 29 or 30) of the pluripotency markers,        mesodermal markers, endodermal markers, neural markers and/or        trophectoderm markers set forth in Table 11;    -   xviii) the relative expression level of each of the markers set        forth in Table 11;    -   xix) any combination of the signatures set forth in xii-xiix;        and/or    -   xx) any combination of the signature set forth in i-xiix;

(j) have the ability to produce a F0 rat;

(k) are capable of being subcultured and maintaining theundifferentiated state;

(l) have the same number of chromosomes as a normal rat cell;

(m) maintain pluripotency in vitro without requiring paracrine LIFsignaling;

(n) have self-renewal, meaning they divide indefinitely whilemaintaining pluripotency;

(o) the rat ES cells express at least one pluripotency marker comprisingDnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor,Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, and/or a combination thereof;

(p) the rat ES cells do not express one or more differentiation markerscomprising c-Myc, Ecat1, and/or Rexo1;

(q) the rat ES cells do not express one or more mesodermal markerscomprising Brachyury, Bmpr2, and/or a combination thereof;

(r) the rat ES cells do not express one or more endodermal markerscomprising Gata6, Sox17, Sox7, and/or combination thereof; and/or

(s) the rat ES cells do not express one or more neural markerscomprising Nestin, Pax6, and/or combination thereof.

One or more of the characteristics outlined in (a)-(s) can be present ina rat ES cell, a rat ES cell population or a rat ES cell line employedin the methods and compositions provided herein, wherein the rat EScells have not undergone a targeted genetic modification. Moreover,following the one or more genetic modification to the rat target locusas described in detail above, the one or more of the characteristicsoutlined in (a)-(s) can be retained in the rat ES cell following thegenetic modification of the target locus.

In one embodiment, the rat ES cell exhibits a homologous recombinationefficiency of at least 2%, at least 3%, at least 4%, at least 5%, atleast 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least11%, at least 12%, at least 13%, at least 14%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, or at least 80%.

In one embodiment, the homologous recombination efficiency employing therat ES cell is greater than 4%.

In one embodiment, the rat ES cell has a doubling time ranging from 24hours to 36 hours. In one embodiment, the rat ES cell has a doublingtime of 25 hours.

In one embodiment, the rat ES cell can be passaged up to at least 15times in 2i medium (Millipore Cat. SF016-200). In one embodiment, therat ES cell can be passaged at least 14 times in 2i medium (MilliporeCat. No. SF016-200). In one embodiment, the rat ES cell can be passagedat least 13, 12, 11, 10, 9, 8, 7, 6, or 5 times in 2i medium.

In one embodiment, when transplanted into a pre-morula stage rat embryo,the rat ES cell can contribute to at least 90% of the cells in an F0generation. In one embodiment, when transplanted into a pre-morula stagerat embryo, the rat ES cell can contribute to at least 95%, 96%, 97%,98%, or 99% of the cells in an F0 generation.

In specific embodiments, the various rat ES cells and cell linesemployed in the various methods and compositions provided herein areused to generate a targeted modification at a target locus. The rat EScell having these targeted genetic modifications can be germ-linecompetent, meaning when the rat ES cell having the targeted geneticmodification is implanted into a rat host embryo, the targeted geneticmodification of the rat ES cell is transmitted to the offspring (i.e.,the F1 population). Thus, in various aspects, the rat ES cells in thevarious methods and compositions are employed to obtain a highfrequency, or high efficiency, of germline transmission of a rat cellgenome from rat ES cells that have undergone a targeted geneticmodification. In various embodiments, the frequency of germlinetransmission is greater than 1:600, greater than 1:500, greater than1:400, greater than 1:300, greater than 1:200, and greater than 1:100.In various embodiments, the frequency of germline transmission isgreater than 1%, greater than 2%, greater than 3%, greater than 4%,greater than 5%, greater than 6%, greater than 7%, greater than 8%,greater than 9%, greater than 10%, up to about 16%, greater than 25%,greater than 50%, greater than 60%, greater than 65%, greater than 70%,greater than 75% or greater. In various embodiments, the frequency ofgermline transmission ranges from 9% to 16%. In various aspects, percentof donor rESC-derived progeny in the F1 generation is 1% or more, 2% ormore, 3% or more, 10% or more, 20% or more, 30% or more, 40% or more,50% or more, 60% or more, from 3% to about 10% or more; from 3% or moreto about 63%, from about 10% to about 30%, from about 10% to about 50%,from about 30% to about 70%, from about 30% to about 60%, from about 20%to about 40%, from about 20% to 65%, or from about 40% to 70%. Thus, arat ES cell that has a targeted genetic modification have the ability totransmit their genome into the F1 population.

A rat ES cell that has a targeted genetic modification can bepluripotent and/or totipotent. Various methods can be used to determineif a rat ES cell is pluripotent. For example, the ES cell can be assayedfor the expression of various pluripotent markers including, but notlimited to, Oct-4, Sox2, alkaline phosphatase, or a combination thereof.See, for example, Okamoto, K. et al., Cell, 60: 461-472 (1990), Scholer,H. R. et al., EMBO J. 9: 2185-2195 (1990)) and Nanog (Mitsui, K. et al.,Cell, 113: 631-642 (2003), Chambers, I. et al., Cell, 113: 643-655(2003) for various methods of assaying for the presence or the level ofsuch markers. See, also FIGS. 2 and 3 provided herein. Otherpluripotency markers include, for example, the presence of at least 1,2, 3, 4, or 5 pluripotency marker comprising Nanog, Klf4, Dppa2, Fgf4,Rex1, Eras, Err-beta and/or Sall3. Other pluripotency markers include,for example, the absence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10pluripotency marker comprising T/Brachyury, Flk1, Nodal, Bmp4, Bmp2,Gata6, Sox17, Hhex1, Sox7, and/or Pax6.

In specific embodiments, the expression and/or the level of expressionof these markers can be determined using RT-PCR. Various kits areavailable to determine the level and/or presence of alkalinephosphatase, including, for example, an ALP tissue staining kit (Sigma)and Vector Red Alkaline Phosphatase Substrate Kit I (Funakoshi) and thelike. Additional assays include in situ hybridization,immunohistochemistry, and immunofluorescence. In specific embodiments,the rat ES cell is characterized by expression of at least onepluripotency marker, including for example expression of Oct-4, Sox2,alkaline phosphatase, or a combination thereof, and preferably all threeof these markers.

The various rat ES cells employed in the method and compositionsprovided herein are capable of maintaining pluripotency and/ortotipotency while being maintained in in vitro culturing conditions.Thus, the various rat ES cells provide herein can, in some embodiments,be subcultured while still maintaining the undifferentiated state.Various methods of culturing the rat ES cells are discussed in furtherdetail elsewhere herein and in US 2014-0235933, herein incorporated byreference in its entirety.

In some embodiments, the rat embryonic stem cells employed herein havebeen isolated from the rat embryo employing various isolation,purification, and culture expansion techniques which are discussed indetail in US 2014-0235933, herein incorporated by reference in itsentirety.

An “isolated” rat ES cell or rat embryo has been removed from itsnatural environment. The term “isolated” can mean free from 70%, 80%,90%, 95%, 96%, 97%, 98% or 99% of the constituents with which acomponent is found in its natural state. As used herein, a rat ES “cellline” comprises a population of isolated rat cells that were developedfrom a single rat ES cell and therefore the population of cells within agiven cell line have a uniform genetic makeup other than for mutationsor karyotypic changes occurring during propagation or during targetedgenetic modifications. For example, rat ES cells can be characterized bya high level of euploidy. Nevertheless, in some cell lines the level ofeuploidy is less than 100% due to karyotypic changes in propagation ofthe line from a single cell. Moreover, a given population of rat EScells can comprise at least 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, or 1×10¹⁰ cells or greater. Some cell populations have sufficientcells to permit selection of a desired modified cell but not anexcessively greater number so as to reduce the possibility of mutationsor karyotypic changes developing in the cell line. For example, somecell populations have 1×10³ to 1×10⁶ cells.

As discussed elsewhere herein, various methods are provided for thetargeted genetic modification of a rat ES cell line. When such methodsare carried out, at least one cell within a rat ES cell line containsthe targeted genetic modification. Through various culturing and/orselection techniques rat ES cell lines having one or more desiredtargeted genetic modifications are produced.

In specific embodiments, a rat ES cell, a population of rat ES cell or arat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) are euploid,and thus have a chromosome number that is an exact multiple of thehaploid number. In further embodiment, a rat ES cell, a population ofrat ES cells or a rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) arediploid, and thus have two haploid sets of homologous chromosomes. Whenreferring to a rat ES cell population or a population of cells from agiven population of rat ES cells or a rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification), at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the cells with the given population are euploid and/or diploid. Inother instances, when referring to a rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification), at least about 50% to 95%, about 60% to 90%, about 60% to95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about 70% to85%, about 70% to about 90%, about 70% to about 95%, about 70% to about100%, about 80% to about 100%, about 80% to about 95%, about 80% toabout 90%, about 90% to about 100%, about 90% to about 99%, about 90% toabout 98%, about 90% to about 97%, about 90% to about 95% of the cellswithin the given population are euploid and/or diploid.

In still further embodiments, a rat ES cell, a population of rat EScells or a rat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) have 42chromosomes. When referring to a rat ES cell population or a populationof cells from a given rat ES cell line (that have not undergone atargeted genetic modification and/or have a targeted geneticmodification) at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%of the cells with the given population have 42 chromosomes. In otherinstances, when referring to a rat ES cell population or a population ofcells from a given rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) atleast about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60% to85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70% toabout 90%, about 70% to about 95%, about 70% to about 100%, about 80% toabout 100%, about 80% to about 95%, about 80% to about 90%, about 90% toabout 100%, about 90% to about 99%, about 90% to about 98%, about 90% toabout 97%, about 90% to about 95% of the cells within the givenpopulation have 42 chromosomes.

In further embodiments, a rat ES cell, a population of rat ES cells or arat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) providedherein form sphere-like colonies when plated on a feeder cell layer invitro. The “sphere-like” morphology refers to the shape of rat ES cellcolonies in culture, rather than the shape of individual ES cells. Therat ES cell colonies are spherical-like. Colonies, which are looselyattached to the feeder cells appear circular (have a circular-likemorphology). Free-floating colonies are spherical-like. The rat ES cellcolonies are spherical-like and very compact, meaning: the boundariesbetween cells are very hard to see. The edge of the colony appearsbright and sharp. Individual nuclei are difficult to distinguish becausethe cells are very small (so that the nucleus takes up most of thevolume of the cell). Mouse ES Cells form elongated colonies and attachstrongly to feeder cells. mESC morphology can vary with strain; e.g. B6colonies are rounder and more domed than F1H4 colonies but are stillmore elongated than rESC. Human ES cell colonies are flatter and morespread out than mESC colonies. The instant rat ES colonies are not flatand do not resemble human ES cell colonies.

In still further embodiments, a rat ES cell, a population of rat EScells or a rat ES cell line (that have not undergone a targeted geneticmodification and/or have a targeted genetic modification) have acircular morphology. A morphology scale for a circle is provided below,where a score of a 10 represents a perfect circle and a score of a 1represents an ellipse.

Morphology Scale of a Circle:

10=A circle with a structure having a longitudinal axis and a verticalaxis that run through the center of the structure and are of equallength.

9=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.9999 to 0.9357 the length of the other axis.

8=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.9357 to 0.875 the length of the other axis.

7=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.875 to about 0.8125 the length of the other axis.

6=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.8125 to 0.750 the length of the other axis.

5=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.750 to 0.6875 the length of the other axis.

4=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.6875 to 0.625 the length of the other axis.

3=A structure having a longitudinal axis and vertical axis that runthrough the center of the structure, wherein one of the axis is between0.625 to 0.5625 the length of the other axis.

2=A structure having a longitudinal axis and vertical axis that runthrough the center of the circle, wherein one of the axis is between0.5625 to 0.523 the length of the other axis.

1=An ellipse is defined as having a longitudinal axis and vertical axisthat run through the center of the structure, wherein one of the axis isbetween 0.523 to 0.500 the length of the other axis.

In one non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the cells with the given population have a circular morphologyscore of a 10, 9 or 8. In other embodiments, the rat ES cell populationor a population of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50% to 95%, about 60% to 90%, about60% to 95%, about 60% to 85%, about 60% to 80%, about 70% to 80%, about70% to 85%, about 70% to about 90%, about 70% to about 95%, about 70% toabout 100%, about 80% to about 100%, about 80% to about 95%, about 80%to about 90%, about 90% to about 100%, about 90% to about 99%, about 90%to about 98%, about 90% to about 97%, about 90% to about 95% of thecells within the given population have a circular morphology score of a10, 9, or 8.

In another non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the cells with the given population have a circular morphologyscore of a 7, 6, 5, 4 or 3. In other non-limiting embodiments, the ratES cell population or a population of cells from a given rat ES cellline (that have not undergone a targeted genetic modification and/orhave a targeted genetic modification) have at least about 50% to 95%,about 60% to 90%, about 60% to 95%, about 60% to 85%, about 60% to 80%,about 70% to 80%, about 70% to 85%, about 70% to about 90%, about 70% toabout 95%, about 70% to about 100%, about 80% to about 100%, about 80%to about 95%, about 80% to about 90%, about 90% to about 100%, about 90%to about 99%, about 90% to about 98%, about 90% to about 97%, about 90%to about 95% of the cells within the given population have a circularmorphology score of a 7, 6, 5, 4, or 3.

In still further embodiments, sphere-like colonies form when the rat EScells (that have not undergone a targeted genetic modification and/orhave a targeted genetic modification) are plated on a feeder cell layerin vitro. A morphology scale for a sphere is provided below, where ascore of a 10 represents a perfect sphere and a score of a 1 representsa three dimensional elliptical structure.

Morphology scale of a sphere-like structure:

10=A sphere is a structure having an X-axis and a Y-axis and a Z-axiseach of which runs through the center of the structure and are of equallength.

9=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.9999 to 0.9357 the length of at least one of the other axes.

8=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.9357 to 0.875 the length of at least one or both of the other axes.

7=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.875 to 0.8125 the length of at least one or both of the other axes.

6=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.8125 to 0.750 the length of at least one or both of the other axes.

5=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is 0.750 to0.6875 the length of at least one or both of the other axes.

4=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is 0.6875to 0.625 the length of at least one or both of the other axes.

3=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.625 to 0.5625 the length of at least one or both of the other axes.

2=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.5625 to 0.523 the length of at least one or both of the other axes.

1=A structure having an X axis and a Y-axis and a Z-axis that runthrough the center of the structure, wherein one of the axis is between0.523 to 0.500 the length of at least one or both of the other axes.

In one non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the colonies that form when the cells are plated on a feedercell layer in vitro have a sphere-like morphology of a 10, 9 or 8. Inother embodiments, the rat ES cell population or a population of cellsfrom a given rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) haveat least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60%to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70%to about 90%, about 70% to about 95%, about 70% to about 100%, about 80%to about 100%, about 80% to about 95%, about 80% to about 90%, about 90%to about 100%, about 90% to about 99%, about 90% to about 98%, about 90%to about 97%, about 90% to about 95% of the colonies that form when thecells are plated on a feeder cell layer in vitro have a sphere-likemorphology of a 10, 9 or 8.

In another non-limiting embodiment, the rat ES cell population or apopulation of cells from a given rat ES cell line (that have notundergone a targeted genetic modification and/or have a targeted geneticmodification) have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% of the colonies that form when the cells are plated on a feedercell layer in vitro have a sphere-like morphology of a 7, 6, 5, 4, or 3.In other embodiments, the rat ES cell population or a population ofcells from a given rat ES cell line (that have not undergone a targetedgenetic modification and/or have a targeted genetic modification) haveat least about 50% to 95%, about 60% to 90%, about 60% to 95%, about 60%to 85%, about 60% to 80%, about 70% to 80%, about 70% to 85%, about 70%to about 90%, about 70% to about 95%, about 70% to about 100%, about 80%to about 100%, about 80% to about 95%, about 80% to about 90%, about 90%to about 100%, about 90% to about 99%, about 90% to about 98%, about 90%to about 97%, about 90% to about 95% of the colonies that form when thecells are plated on a feeder cell layer in vitro have a sphere-likemorphology of a 7, 6, 5, 4, or 3.

A given rat ES cell, employed in the various methods and compositionsprovided herein can be a male (XY) rat ES cell, a male (XY) populationof rat ES cells, or a male (XY) rat ES cell line. In other embodiments,a population of rat ES cells or a rat ES cell line employed herein canbe a female (XX) rat ES cell, a female (XX) population of rat ES cells,or a female (XX) rat ES cell line. Any such rat ES cell, population ofrat ES cells or rat ES cell line can comprise the euploidy and/ordiploidy as described above.

The various rat ES cells employed in the methods and compositions can befrom any rat strain, including but not limited to, an ACI rat strain, aDark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, aSprague Dawley (SD) rat strain, or a Fischer rat strain such as FisherF344 or Fisher F6. The various rat ES cells can also be obtained from astrain derived from a mix of two or more strains recited above. In oneembodiment, the rat ES cell is derived from a strain selected from a DAstrain and an ACI strain. In a specific embodiment, the rat ES cell isderived from an ACI strain. The ACI rat strain is are characterized ashaving black agouti, with white belly and feet and an RT1av1 haplotype.Such strains are available from a variety of sources including HarlanLaboratories. In other embodiments, the various rat ES cells are from aDark Agouti (DA) rat strain, which is characterized as having an agouticoat and an RT1av1 haplotype. Such rats are available from a variety ofsource including Charles River and Harlan Laboratories. In a furtherembodiment, the various rat ES cells employed herein are from an inbredrat strain.

In specific embodiments the rat ES cell line is from an ACI rat andcomprises the ACI.G1 rat ES cell as described in detail in US2014-0235933, herein incorporated by reference in its entirety. Inanother embodiment, the rat ES cell line is from a DA rat and comprisesthe DA.2B rat ES cell line or the DA.2C rat ES cell line as described indetail in US 2014-0235933, herein incorporated by reference in itsentirety.

A given rat ES cell provided herein can be obtained from a rat embryo atvarious stages of rat embryo development. The rat embryos employed toderive the rat ES cells can be a morula-stage embryo, a blastocyst-stageembryo, or a rat embryo at a developmental stage between a morula-stageembryo and a blastocyst-stage embryo. Thus, in specific embodiments, therat embryo employed is at or between the Witschi stages of 5 and 7. Inother embodiments, the rat embryo employed is at the Witschi stage 5, 6,or 7.

In one embodiment, the rat ES cell is obtained from a rat blastocyst. Inother embodiments, the rat ES cell is obtained from a blastocyst from asuperovulated rat. In other embodiments, the rat ES cells are obtainedfrom an 8-cell stage embryo, which is then cultured in vitro until itdevelops into a morula-stage, blastocyst stage, an embryo between theWitschi stages 5 and 7, or into an embryo at the Witschi stage 5, 6, or7. At which time the embryos are then plated. Morula-stage embryoscomprise a compact ball of cells with no internal cavity.Blastocyst-stage embryos have a visible internal cavity (the blastocoel)and contain an inner cell mass (ICM). The ICM cells form ES cells.

B. Derivation and Propagation of Rat Embryonic Stem (ES) Cells

Methods of derivation and propagation of rat embryonic stem cells areknown in the art and are disclosed, for example, in US 2014-0235933,herein incorporated by reference in its entirety. In specificembodiments, such methods comprise (a) providing an in vitro culturecomprising a feeder cell layer and a population of isolated ratembryonic stem (ES) cells; (b) culturing in vitro under conditions whichare sufficient to maintain pluripotency and/or totipotency of theisolated rat ES cell. Such methods thereby allow for the propagation ofa rat ES cell population and/or a rat ES cell line.

Methods for culturing a rat embryonic stem cell line is provided. Suchmethods comprise culturing in vitro a feeder cell layer and a rat EScell line, wherein the culture conditions maintain pluripotency of therat ES cells and comprise a media having mouse leukemia inhibitoryfactor (LIF) or an active variant or fragment thereof. The methods canfurther comprise passaging and culturing in vitro the cells of the ratES cell line, wherein each subsequent in vitro culturing comprisesculturing the rat ES cells on the feeder cell layer under conditionsthat maintain pluripotency of the rat ES cells and comprises a mediahaving mouse LIF or an active variant or fragment thereof.

The culture media employed in the various methods and compositions canmaintain the rat ES cells. The terms “maintaining” and “maintenance”refer to the stable preservation of at least one or more of thecharacteristics or phenotypes of the rat ES cells outline herein. Suchphenotypes can include maintaining pluripotency and/or totipotency, cellmorphology, gene expression profiles and the other functionalcharacteristics of the rat stem cells described herein. The term“maintain” can also encompass the propagation of stem cells, or anincrease in the number of stem cells being cultured. The term furthercontemplates culture conditions that permit the stem cells to remainpluripotent, while the stem cells may or may not continue to divide andincrease in number.

The term “feeder cell” or “feeder cell layer” comprises a culture ofcells that grow in vitro and secrete at least one factor into theculture medium that is used to support the growth of another cell ofinterest in the culture. The feeder cells employed herein aid inmaintaining the pluripotency of the rat ES cells, and in specificembodiments, one or more of the other characteristics or phenotypesdescribed herein. Various feeder cells can be used including, forexample, mouse embryonic fibroblasts, including mouse embryonicfibroblasts obtained between the 12^(th) and 16^(th) day of pregnancy.In specific embodiments, feeder cell layer comprises a monolayer ofmitotically inactivated mouse embryonic fibroblasts (MEFs).

The in vitro cultures of the rat ES cells further comprise an effectiveamount of Leukemia Inhibitory Factor (LIF) or an active variant orfragment thereof. Leukemia inhibitory factor (LIF) belongs to the IL-6receptor family. LIF binds to a heterodimeric membrane receptor made upof a LIF-specific subunit, gp190 or LIFR, and the subunit gp130, whichis shared with the other members of the IL-6 family. LIF inhibits thedifferentiation of embryonic stem cells in mice and contribute to stemcell self-renewal. Human and mouse LIF share 79% sequence homology andexhibit cross-species activity. Rat LIF (rtLIF) is a 22.1 kDa proteincontaining 202 amino acid residues that exhibits 91% amino acid sequenceidentity with murine LIF (Takahama et al. 1998). There are six possibleasparagine-linked glycosylation (N-glycosylation) sites which areconserved among the LIF polypeptide from the various species and anadditional site of Asn150 which is specific for rat LIF. The tertiarystructure of the mouse LIF and its function is described in furtherdetail in Aikawa et al. (1998) Biosci. Biotechnol. Biochem. 62 1318-1325and Senturk et al. (2005) Immunology of Pregnancy, editor Gil Mor., U.S.Pat. No. 5,750,654 and D P Gearing (1987) EMBO Journal 1987-12-20, eachof which is herein incorporated by reference in their entirety. Apartial mouse LIF sequence is reported on the SwissProt website underthe accession number P09056.

Mouse LIF activity is assessed by its ability to induce differentiationof M1 myeloid leukemia cells. The specific activity is 1×10⁶ units/ml(Cat. No. 03-0011 from Stemgent) and 1×10⁷ units/ml (Cat. No.03-0011-100 from Stemgent), where 50 units is defined as the amount ofmouse LIF required to induce differentiation in 50% of the M1 coloniesin 1 ml of medium. See, also, Williams, R. L. et al. (1988) Nature 336:684-687.; Metcalf, D. et al. (1988) Leukemia 2: 216-221; Niwa, H. et al.(2009) Nature 460: 118-122; Xu, J. et al. (2010) Cell Biol Int. 34:791-797; Fukunaga, N. et al. (2010) Cell Reprogram. 12: 369-376; and,Metcalf D. (2003) Stem Cells 21: 5-14, each of which is hereinincorporated by reference in their entirety. An “effective amount ofLIF” comprises a concentration of LIF that allows the rat ES cells of anin vitro culture to remain in an undifferentiated pluripotent state.Various markers that can be used to assay for the cells remaining in apluripotent state are discussed elsewhere herein.

The LIF polypeptide employed in the various methods and compositionsprovided herein can be from any organism, including from a mammal, arodent, a human, a rat or a mouse. In one embodiment, the LIFpolypeptide is from a mouse. In still further embodiments, the mouse LIFpolypeptide comprises the amino acid sequence set forth in SwissProtAccession number: P09056, which is herein incorporated by reference inits entirety and is also set forth in SEQ ID NO: 9.

In other embodiments, an active variant or fragment of the mouse LIFpolypeptide as set forth in SEQ ID NO: 9 or in SwissProt Accessionnumber: P09056 can be used. Such active variants and fragments(including active variants having at least 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9are discussed in further detail elsewhere herein.

LIF polypeptide or the active variant or fragment thereof can beprovided to the in vitro culture in a variety of ways. In oneembodiment, the effective amount of the LIF polypeptide or the activevariant or fragment thereof is added to the culture media. In otherembodiments, the feeder cells have been genetically modified tooverexpress the LIF polypeptide or the active variant or fragmentthereof. Such feeder cells include feeder cells prepared fromgamma-irradiated or mitomycin-C treated DIA-M mouse fibroblasts thatexpress matrix-associated LIF. Method of generating and using suchgenetically modified feeder cells can be found, for example, in See,Buehr et al. (2003) Biol Reprod 68:222-229, Rathjen et al. (1990) Cell62 1105-1115, and Buehr et al. (2008) Cell 135:1287-1298, each of whichis herein incorporated by reference. The heterologous LIF expressed inthe feeder cells can be from the same organism as the feeder cells orfrom an organism that is different from that of the feeder cell. Inaddition, the heterologous LIF expressed in the feeder cells can be fromthe same or from a different organism than the ES cells the feeder layeris supporting.

In still other embodiments, the feeder cells employed in the variousmethods disclosed herein are not genetically modified to express aheterologous LIF polypeptide or an active variant or fragment thereof.Thus, in particular embodiments, the monolayer of mitoticallyinactivated mouse embryonic fibroblast employed in the methods has notbeen genetically modified to express a heterologous LIF polypeptide.

In other embodiments, the LIF polypeptide or the active variant orfragment thereof is added to the culture media. When LIF is added to theculture media, the LIF can be from any organism, including from amammal, a rodent, a human, a rat or a mouse. In one embodiment, the LIFpresent in the culture media is from a mouse. In still furtherembodiments, the mouse LIF polypeptide comprises the amino acid sequenceset forth in SEQ ID NO:9. In other embodiments, an active variant orfragment of the mouse LIF polypeptide as set forth in SEQ ID NO:9 can beused. Such active variants and fragments (including active variantshaving at least 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to SEQ ID NO: 9) are discussed in furtherdetail elsewhere herein.

In specific embodiments, the rat ES cells and rat ES cell lines providedherein maintain pluripotency in vitro without requiring paracrine LIFsignaling.

In specific embodiments, LIF or an active variant or fragment thereof ispresent in the culture media at any concentration that maintains the ratES cells. LIF polypeptide or active variant or fragment thereof ispresent in the culture media at about 25 U/ml to about 50 U/ml, at about50 U/ml to about 100 U/ml, at about 100 U/ml to about 125 U/ml, at about125 U/ml to about 150 U/ml, at about 150 U/ml to about 175 U/ml, atabout 175 U/ml to about 200 U/ml, at about 200 U/ml to about 225 U/ml,at about 225 U/ml to about 250 U/ml, at about 250 U/ml to about 300U/ml, to about 300 U/ml to about 325 U/ml, at about 325 U/ml to about350 U/ml, at about 350 U/ml to about 400 U/ml, at about 400 U/ml toabout 425 U/ml, at about 425 U/ml to about 450 U/ml, at about 450 U/mlto about 475 U/ml, at about 475 U/ml to about 500 U/ml, at about 75 U/mlto about 500 U/ml or greater. In other embodiments, LIF polypeptide oractive variant or fragment thereof is present in the culture media atabout 25 U/ml to about 50 U/ml, at about 25 U/ml to about 100 U/ml, atabout 75 U/ml to about 125 U/ml, at about 50 U/ml to about 150 U/ml, atabout 90 U/ml to about 125 U/ml, at about 90 U/ml to about 110 U/ml, atabout 80 U/ml to about 150 U/ml, or at about 80 U/ml to about 125 U/ml.In a specific embodiment, LIF polypeptide or active variant or fragmentthereof is present in the culture media at about 100 U/ml.

When mouse LIF is employed, the mouse LIF polypeptide or active variantor fragment thereof is present in the culture media at any concentrationthat maintains the rat ES cells. Mouse LIF polypeptide or active variantor fragment thereof is present at about 25 U/ml to about 50 U/ml, atabout 50 U/ml to about 100 U/ml, at about 100 U/ml to about 125 U/ml, atabout 125 U/ml to about 150 U/ml, at about 150 U/ml to about 175 U/ml,at about 175 U/ml to about 200 U/ml, at about 200 U/ml to about 225U/ml, at about 225 U/ml to about 250 U/ml, at about 250 U/ml to about300 U/ml, to about 300 U/ml to about 325 U/ml, at about 325 U/ml toabout 350 U/ml, at about 350 U/ml to about 400 U/ml, at about 400 U/mlto about 425 U/ml, at about 425 U/ml to about 450 U/ml, at about 450U/ml to about 475 U/ml, at about 475 U/ml to about 500 U/ml, at about 75U/ml to about 500 U/ml or greater. In other embodiments, mouse LIFpolypeptide or active variant or fragment thereof is present at about 25U/ml to about 50 U/ml, at about 25 U/ml to about 100 U/ml, at about 75U/ml to about 125 U/ml, at about 50 U/ml to about 150 U/ml, at about 90U/ml to about 125 U/ml, at about 90 U/ml to about 110 U/ml, at about 80U/ml to about 150 U/ml, or at about 80 U/ml to about 125 U/ml. In aspecific embodiment, mouse LIF polypeptide or active variant or fragmentthereof is present in the culture media at about 100 U/ml.

The culture media employed maintains rat ES cells. As such, in specificembodiments, the culture media employed in the various method andcompositions will maintain the pluripotency of all or most of (i.e.,over 50%) of the rat ES cells in a cell line for a period of a at least5, 10 or 15 passages. In one embodiment, the culture media comprises oneor more compounds that assist in maintaining pluripotency. In oneembodiment, the culture media comprises a MEK pathway inhibitor and aglycogen synthase kinase-3 (GSK-3) inhibitor. The media can furthercomprise additional components that aid in maintaining the ES cells,including for example, FGF receptor inhibitors, ROCK inhibitors, and/orALK (TGFb receptor) inhibitors. A non-limiting example of an FGFreceptor inhibitors includes PD184352. A non-limiting example of a ROCKinhibitor includes Y-27632, and non-limiting example of an ALK (TGFbreceptor) inhibitor includes A-83-01. In specific embodiments, 2i mediais used with 10 uM ROCKi when thawing cryopreserved rESC or whenre-plating rESC after dissociation with trypsin.

In other embodiments, the media comprises a combination of inhibitorsconsisting of a MEK pathway inhibitor and a glycogen synthase kinase-3(GSK-3) inhibitor.

In one non-limiting embodiment, the culture media comprises a GSK-3inhibitor comprising CHIR99021 and/or comprises a MEK inhibitorcomprising PD0325901. In other embodiments, the media comprises acombination of inhibitors consisting of CHIR99021 and PD0325901. Eitherof these compounds can be obtained, for example, from Stemgent. Inspecific embodiments, CHIR99021 is present in the culture media at aconcentration of about 0.5μ to about 3 μM, about 0.5μ to about 3.5 μM,about 0.5 μM to about 4 μM, about 0.5 μM to about 1 μM, about 1 μM toabout 1.5 μM, about 1.5 μM to about 2 μM, about 2 μM to about 2.5 μM,about 2.5 to about 3 μM, 3 μM to about 3.5 μM. In further embodiments,CHIR99021 is present in the culture media at a concentration of about 3μM. In other embodiments, PD0325901 is present in the culture media at aconcentration of about 0.4 μM to about 1 uM, about 0.4 μM to about 1.5uM, about 0.4 μM to about 2 μM, about 0.4 μM to about 0.8 μM, 0.8 μM toabout 1.2 μM, about 1.2 to about 1.5 μM. In further embodiments,PD0325901 is present in the culture media at a concentration of about 1μM. In specific embodiments, CHIR99021 is present in the culture mediaat a concentration of about 3 μM and PD0325901 is present at aconcentration of about 1 μM.

In one non-limiting embodiment, the culture media employed in thevarious methods and compositions disclosed herein is a 2i media whichcomprises: DMEM/F12 basal media (at a concentration of 1× (50%));Neurobasal media (at a concentration of 1× (50%));Penicillin/streptomycin (at a concentration of 1%); L-Glutamine (at aconcentration of 4 mM); 2-Mercaptoethanol (at a concentration of 0.1mM); N2 supplement (at a concentration of 1×); B27 supplement (at aconcentration 1×); LIF (at a concentration of 100 U/ml); PD0325901 (MEKinhibitor) (at a concentration of 1 μM) and CHIR99021 (GSK inhibitor)(at a concentration of 3 μM).

Additional media that can be employed include those disclosed in Li etal. (2008) Cell 135:1299-1310, Yamamoto et al. (2012) Transgenic Rats21:743-755, Ueda et al. (2008) PLoS ONE 3(6):e2800, Meek et al. (2010)PLoS ONE 4 (12): e14225; Tong et al. (2010) Nature 467:211-213; USPatent Publication 2012/0142092, Buehr et al. (2008) Cell 135:1287-1298,Li et al. (135) Cell 1299-1310, each of which is herein incorporated byreference in their entirety. When employing such media, theconcentration and the source of LIF can be modified as outlined herein.In specific embodiments, the various culture medias are used incombination with mouse LIF or an active variant or fragment thereof, andin even further embodiments, the various culture medias comprise a mouseLIF or an active variant or fragment thereof at a concentration of about50 U/ml to about 100 U/ml, about 50 U/ml to about 150 U/ml, or about 100U/ml.

The temperature of the cultures of rat ES cells, both for the productionof the ES cell line and for the culturing and maintaining of the ES lineit typically carried out at about 35° C. to about 37.5° C. In specificembodiment, the temperature is 37.0° C. The culture is typically carriedout at 7.5% CO₂.

7. Sequence Identity

The methods and compositions provided herein employ a variety ofdifferent components of the targeted genomic integration system (i.e.nuclease agents, recognition sites, insert nucleic acids,polynucleotides of interest, targeting vectors, selection markers andother components). It is recognized throughout the description that somecomponents of the targeted genomic integration system can have activevariants and fragments. Such components include, for example, nucleaseagents (i.e. engineered nuclease agents), nuclease agent recognitionsites, polynucleotides of interest, target sites and correspondinghomology arms of the targeting vector. Biological activity for each ofthese components is described elsewhere herein.

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof “Equivalentprogram” means any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein also can beused in the practice or testing of the described invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the described inventionis not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates, which may need to be independentlyconfirmed.

The described invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indicating the scope of theinvention.

Non-limiting embodiments include:

1. A method for targeted modification of a genomic locus of interest ina pluripotent rat cell, comprising (a) introducing into the pluripotentrat cell a large targeting vector (LTVEC) comprising an insert nucleicacid flanked with a 5′ rat homology arm and a 3′ rat homology arm,wherein the sum total of the 5′ and the 3′ homology arms is at least 10kb but less than 150 kb; and (b) identifying a genetically modifiedpluripotent rat cell comprising the targeted genetic modification at thegenomic locus of interest, wherein the targeted genetic modification iscapable of being transmitted through the germline.

2. The method of embodiment 1, wherein the targeted genetic modificationis biallelic.

3. The method of embodiment 1 or 2, wherein the pluripotent rat cell isa rat embryonic stem (ES) cell.

4. The method of embodiment 1, 2 or 3, wherein the pluripotent rat cellis derived from a DA strain or an ACI strain.

5. The method of any one of embodiments 1-4, wherein the pluripotent ratcell is characterized by expression of at least one pluripotency markercomprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIFreceptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or a combinationthereof.

6. The method of any one of embodiments 1-4 wherein the pluripotent ratcell is characterized by one of more of the following characteristics:

(a) lack of expression of one or more pluripotency markers comprisingc-Myc, Ecat1, and/or Rexo1; (b) lack of expression of mesodermal markerscomprising Brachyury and/or Bmpr2; (c) lack of expression of one or moreendodermal markers comprising Gata6, Sox17 and/or Sox7; or (d) lack ofexpression of one or more neural markers comprising Nestin and/or Pax6.

7. The method of any one of embodiments 1-6, wherein the sum total ofthe 5′ and the 3′ homology arms of the LTVEC is from about 10 kb toabout 30 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb.

8. The method of any one of embodiments 1-6, wherein the sum total ofthe 5′ and the 3′ homology arms of the LTVEC is from about 16 Kb toabout 150 Kb.

9. The method of any one of embodiments 1-8, wherein the targetedgenetic modification comprises: (a) a replacement of an endogenous ratnucleic acid sequence with a homologous or an orthologous nucleic acidsequence; (b) a deletion of an endogenous rat nucleic acid sequence; (c)a deletion of an endogenous rat nucleic acid sequence, wherein thedeletion ranges from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200kb, from about 200 kb to about 300 kb, from about 300 kb to about 400kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb,from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, fromabout 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (d) anexogenous nucleic acid sequence ranging from about 5 kb to about 10 kb,from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, fromabout 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kbto about 300 kb, from about 300 kb to about 350 kb, or from about 350 kbto about 400 kb; (e) an exogenous nucleic acid sequence comprising ahomologous or an orthologous nucleic acid sequence; (f) a chimericnucleic acid sequence comprising a human and a rat nucleic acidsequence; (g) a conditional allele flanked with site-specificrecombinase target sequences; or, (h) a reporter gene operably linked toa promoter active in a rat cell.

10. The method of any one of embodiments 1-9, wherein the genomic locusof interest comprises (i) a first nucleic acid sequence that iscomplementary to the 5′ rat homology arm; and (ii) a second nucleic acidsequence that is complementary to the 3′ rat homology arm.

11. The method of embodiment 10, wherein the first and the secondnucleic acid sequence is separated by at least 5 kb but less than 3 Mb.

12. The method of embodiment 10, wherein the first and the secondnucleic acid sequence is separated by at least 5 kb but less than 10 kb,at least 10 kb but less than 20 kb, at least 20 kb but less than 40 kb,at least 40 kb but less than 60 kb, at least 60 kb but less than 80 kb,at least about 80 kb but less than 100 kb, at least 100 kb but less than150 kb, or at least 150 kb but less than 200 kb, at least about 200 kbbut less than about 300 kb, at least about 300 kb but less than about400 kb, at least about 400 kb but less than about 500 kb, at least about500 kb but less than about 1 Mb, at least about 1 Mb but less than about1.5 Mb, at least about 1.5 Mb but less than about 2 Mb, at least about 2Mb but less than about 2.5 Mb, or at least about 2.5 Mb but less thanabout 3 Mb.

13. The method of any one of embodiment 1-12, wherein introducing step(a) further comprises introducing a second nucleic acid encoding anuclease agent that promotes a homologous recombination between thetargeting construct and the genomic locus of interest in the pluripotentrat cell.

14. The method of embodiment 13, wherein the nuclease agent comprises(a) a chimeric protein comprising a zinc finger-based DNA binding domainfused to a FokI endonuclease; or, (b) a chimeric protein comprising aTranscription Activator-Like Effector Nuclease (TALEN) fused to a FokIendonuclease.

15. The method of any one of embodiments 1-12, wherein introducing step(a) further comprises introducing into the pluripotent rat cell: (i) afirst expression construct comprising a first promoter operably linkedto a first nucleic acid sequence encoding a Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein,(ii) a second expression construct comprising a second promoter operablylinked to a genomic target sequence linked to a guide RNA (gRNA),wherein the genomic target sequence is immediately flanked on the 3′ endby a Protospacer Adjacent Motif (PAM) sequence.

16. The method of embodiment 15, wherein the genomic locus of interestcomprises the nucleotide sequence of SEQ ID NO: 1.

17. The method of embodiment 15 or 16, wherein the gRNA comprises athird nucleic acid sequence encoding a Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activatingCRISPR RNA (tracrRNA).

18. The method of embodiment 15, 16 or 17, wherein the Cas protein isCas9.

19. The method of embodiment 15, 16, 17, or 18, wherein the gRNAcomprises: (a) the chimeric RNA of the nucleic acid sequence of SEQ IDNO: 2; or, (b) the chimeric RNA of the nucleic acid sequence of SEQ IDNO: 3.

20. The method of embodiment 17, wherein the crRNA comprises SEQ ID NO:4; SEQ ID NO: 5; or SEQ ID NO: 6.

21. The method of embodiment 17, wherein the tracrRNA comprises SEQ IDNO: 7 or SEQ ID NO: 8.

22. A modified rat genomic locus comprising: (i) an insertion of ahomologous or orthologous human nucleic acid sequence; (ii) areplacement of an endogenous rat nucleic acid sequence with thehomologous or orthologous human nucleic acid sequence; or (iii) acombination thereof, wherein the modified rat genomic locus is capableof being transmitted through the germline.

23. The modified rat genomic locus of embodiment 22, wherein the size ofthe insertion or replacement is from about 5 kb to about 400 kb.

24. The rat genomic locus of embodiment 22, wherein the size of theinsertion or replacement is from about 5 kb to about 10 kb, from about10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kbto about 60 kb, from about 60 kb to about 80 kb, from about 80 kb toabout 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, or from about 350 kb toabout 400 kb.

25. A method for making a humanized rat, comprising: (a) targeting agenomic locus of interest in a pluripotent rat cell with a targetingconstruct comprising a human nucleic acid to form a genetically modifiedpluripotent rat cell; (b) introducing the genetically modifiedpluripotent rat cell into a host rat embryo; and (c) gestating the hostrat embryo in a surrogate mother; wherein the surrogate mother producesrat progeny comprising a modified genomic locus that comprises: (i) aninsertion of a human nucleic acid sequence; (ii) a replacement of therat nucleic acid sequence at the genomic locus of interest with ahomologous or orthologous human nucleic acid sequence; (iii) a chimericnucleic acid sequence comprising a human and a rat nucleic acidsequence; or (iv) a combination thereof, wherein the modified genomiclocus is capable of being transmitted through the germline.

26. The method of embodiment 25, wherein the targeting construct is alarge targeting vector (LTVEC), and the sum total of the 5′ and the 3′homology arms of the LTVEC is at least 10 kb but less than 150 kb.

27. The method of embodiment 26, wherein the sum total of the 5′ and the3′ homology arms of the targeting construct is from about 10 kb to about30 kb, from about 20 kb to 40 kb, from about 40 kb to about 60 kb, fromabout 60 kb to about 80 kb, or from about 80 kb to about 100 kb, fromabout 100 kb to about 120 kb, or from about 120 kb to 150 kb.

28. The method of embodiment 25, 26 or 27, wherein the human nucleicacid sequence is at least 5 kb but less than 400 kb.

29. The method of embodiment 25, 26, or 27, wherein the human nucleicacid sequence is at least 5 kb but less than 10 kb, at least 10 kb butless than 20 kb, at least 20 kb but less than 40 kb, at least 40 kb butless than 60 kb, at least 60 kb but less than 80 kb, at least about 80kb but less than 100 kb, at least 100 kb but less than 150 kb, at least150 kb but less than 200 kb, at least 200 kb but less than 250 kb, atleast 250 kb but less than 300 kb, at least 300 kb but less than 350 kb,or at least 350 kb but less than 400 kb.

30. The method of any one of embodiments 25-29, wherein the pluripotentrat cell is a rat embryonic stem (ES) cell.

31. The method of any one of embodiments 25-30, wherein the pluripotentrat cell is derived from a DA strain or an ACI strain.

32. The method of any one of embodiments 25-31, wherein the pluripotentrat cell is characterized by expression of at least one pluripotencymarker comprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4,Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, or acombination thereof.

33. The method of any one of embodiment 25-31, wherein the pluripotentrat cell is characterized by one or more of the following features: (a)lack of expression of one or more pluripotency markers comprising c-Myc,Ecat1, and/or Rexo1; (b) lack of expression of one or more mesodermalmarkers comprising Brachyury and/or Bmpr2; (c) lack of expression of oneor more endodermal markers comprising Gata6, Sox17, and/or Sox7; or (d)lack of expression of one or more neural markers comprising Nestinand/or Pax6.

34. A modified rat comprising a humanized genomic locus, wherein thehumanized genomic locus comprises: (i) an insertion of a homologous ororthologous human nucleic acid sequence; (ii) a replacement of a ratnucleic acid sequence at an endogenous genomic locus with a homologousor orthologous human nucleic acid sequence; (iii) a chimeric nucleicacid sequence comprising a human and a rat nucleic acid sequence or,(iv) a combination thereof, wherein the humanized genomic locus iscapable of being transmitted through the germline.

35. A rat or rat cell comprising a targeted genetic modification in itsgenomic locus, wherein the genomic locus is an Interleukin-2 receptorgamma locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or a Rag2/Rag1locus, wherein the targeted genetic modification comprises: (a) adeletion of an endogenous rat nucleic acid sequence at the genomiclocus; (b) an insertion of a homologous nucleic acid, an orthologousnucleic acid, or a chimeric nucleic acid comprising a human and a ratnucleic acid sequence, or (c) a combination thereof, wherein thetargeted genetic modification is transmissible through the germline ofthe rat or a rat propagated from the rat cell.

36. The rat or rat cell of embodiment 35, wherein (a) the deletion ofthe endogenous rat nucleic acid at the genomic locus is at least about10 kb; or, (b) the deletion of the endogenous rat nucleic acid at thegenomic locus is from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200kb, from about 200 kb to about 300 kb, from about 300 kb to about 400kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb,from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, fromabout 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (c) theinsertion of the exogenous nucleic acid sequence at the genomic locus isat least about 5 kb; or, (d) the insertion of the exogenous nucleic acidsequence at the genomic locus is from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, or from about 350 kb toabout 400 kb.

37. The rat or rat cell of embodiment 35 or 36, wherein (a) the targetedgenetic modification at the Interleukin-2 receptor gamma locus resultsin a decrease in or absence of Interleukin-2 receptor gamma proteinactivity; (b) the targeted genetic modification at the ApoE locusresults in a decrease in or absence of ApoE protein activity; (c) thetargeted genetic modification at the Rag1 locus results in a decrease inor absence of Rag1 protein activity; (d) the targeted geneticmodification at the Rag2 locus results in a decrease in or absence ofRag2 protein activity; or, (e) the targeted genetic modification at theRag2/Rag1 locus results in a decrease in or absence of Rag2 proteinactivity and Rag1 activity.

38. The rat or rat cell of embodiment 35, 36, or 37, wherein thetargeted genetic modification of the Interleukin-2 receptor gamma locuscomprises: (a) a deletion of the entire rat Interleukin-2 receptor gammacoding region or a portion thereof; (b) a replacement of the entire ratInterleukin-2 receptor gamma coding region or a portion thereof with ahuman Interleukin-2 receptor gamma coding region or a portion thereof;(c) a replacement of an ecto-domain of the rat Interleukin-2 receptorgamma coding region with the ecto-domain of a human Interleukin-2receptor gamma; or, (d) at least a 3 kb deletion of the Interleukin-2receptor gamma locus.

39. The rat or rat cell of any one of embodiments 35-37, wherein thetargeted genetic modification of the ApoE locus comprises: (a) adeletion of the entire ApoE coding region or a portion thereof; or, (b)at least a 1.8 kb deletion of the ApoE locus comprising the ApoE codingregion.

40. The rat or rat cell of any one of embodiments 35-37, wherein thetargeted genetic modification of the Rag2 locus comprises: (a) adeletion of the entire Rag2 coding region or a portion thereof; (b) atleast a 5.7 kb deletion of the Rag2 locus comprising the Rag2 codingregion.

41. The rat or rat cell of any one of embodiments 35-37, wherein thetargeted genetic modification of the Rag2/Rag1 locus comprises: (a) adeletion of the entire Rag2 coding region or a portion thereof and adeletion of the entire Rag1 coding region or portion thereof; or, (b) adeletion of at least 16 kb of the Rag2/Rag1 locus comprising the Rag2coding region.

42. The rat or rat cell of any one of embodiment 35-41, wherein thetargeted genetic modification comprises an insertion of an expressioncassette comprising a selective marker at the Interleukin-2 receptorgamma locus, the ApoE locus, the Rag1 locus, the Rag2 locus, or theRag2/Rag1 locus.

43. The rat or rat cell of any one of embodiments 42, wherein theexpression cassette comprises a lacZ gene operably linked to theendogenous promoter at the genomic locus and a human ubiquitin promoteroperably linked to a selective marker.

44. The rat or rat cell of any one of embodiments 35-43, wherein thetargeted genetic modification in the Interleukin-2 receptor gamma locus,the ApoE locus, the Rag1 locus, the Rag2 locus or the Rag2/Rag1 locuscomprises the insertion of a self-deleting selection cassette.

45. The rat or rat cell of embodiment 44, wherein the self-deletingselection cassette comprises a selective marker gene operably linked toa promoter active in the rat cell and a recombinase gene operably linkedto a male germ cell-specific promoter, wherein the self-deletingcassette is flanked by recombination recognition sites recognized by therecombinase.

46. The rat or rat cell of embodiment 45, wherein (a) the male germcell-specific promoter is a Protamine-1 promoter; or, (b) therecombinase gene encodes Cre, and the recombination recognition sitesare loxP sites.

47. The rat or rat cell of any one of embodiments 35-46, wherein theinsertion of the exogenous nucleic acid sequence at the genomic locuscomprises a reporter nucleic acid operably linked to an endogenousInterleukin-2 receptor gamma promoter, an endogenous ApoE promoter, anendogenous Rag1 promoter, or an endogenous Rag2 promoter.

48. The rat or rat cell of embodiment 47, wherein the reporter nucleicacid encodes a reporter comprising β-galactosidase, mPlum, mCherry,tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus,YPet, enhanced yellow fluorescent protein (EYFP), Emerald, enhancedgreen fluorescent protein (EGFP), CyPet, cyan fluorescent protein (CFP),Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combinationthereof.

49. The rat cell of any one of embodiments 35-48, wherein the rat cellis a pluripotent rat cell or a rat embryonic stem (ES) cell.

50. The rat cell of embodiment 49, wherein the pluripotent rat cell orthe rat embryonic stem (ES) cell (a) is derived from a DA strain or anACI strain; (b) is characterized by expression of at least onepluripotency marker comprising Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4,Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1,or a combination thereof; or (c) is characterized by one or more of thefollowing characteristics: (i) lack of expression of one or morepluripotency markers comprising c-Myc, Ecat1, and/or Rexo1; (ii) lack ofexpression of mesodermal markers comprising Brachyury and/or Bmpr2;(iii) lack of expression of one or more endodermal markers comprisingGata6, Sox17 and/or Sox7; or (iv) lack of expression of one or moreneural markers comprising Nestin and/or Pax6.

51. A method for modifying a target genomic locus in an Interleukin-2receptor gamma locus, an ApoE locus, a Rag1 locus, a Rag2 locus or aRag2/Rag1 locus in a pluripotent rat cell, the method comprising: (a)introducing into the pluripotent rat cell a targeting vector comprisingan insert nucleic acid flanked with 5′ and 3′ rat homology armshomologous to the target genomic locus, (b) identifying a geneticallymodified pluripotent rat cell comprising a targeted genetic modificationat the target genomic locus, wherein the targeted genetic modificationis capable of being transmitted through the germline of a rat propagatedfrom the pluripotent rat cell.

52. The method of embodiment 51, wherein the targeting vector is a largetargeting vector (LTVEC) wherein the sum total of the 5′ and the 3′ rathomology arms is at least about 10 kb but less than about 150 kb.

53. The method of embodiment 51 or 52, wherein introducing the targetingvector into the pluripotent rat cell leads to: (i) a deletion of anendogenous rat nucleic acid sequence at the target genomic locus; (ii)an insertion of an exogenous nucleic acid sequence at the target genomiclocus; or (iii) a combination thereof.

54. The method of embodiment 53, wherein (a) the deletion of theendogenous rat nucleic acid at the genomic locus is at least about 10kb; or, (b) the deletion of the endogenous rat nucleic acid at thegenomic locus is from about 5 kb to about 10 kb, from about 10 kb toabout 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200kb, from about 200 kb to about 300 kb, from about 300 kb to about 400kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb,from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, fromabout 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb; (c) theinsertion of the exogenous nucleic acid sequence at the genomic locus isat least about 5 kb; or. (d) the insertion of the exogenous nucleic acidsequence at the genomic locus is from about 5 kb to about 10 kb, fromabout 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kbto about 100 kb, from about 100 kb to about 150 kb, from about 150 kb toabout 200 kb, from about 200 kb to about 250 kb, from about 250 kb toabout 300 kb, from about 300 kb to about 350 kb, or from about 350 kb toabout 400 kb.

55. The method of any one of embodiment 51-54, wherein (a) the targetedgenetic modification at the Interleukin-2 receptor gamma locus resultsin a decrease in or absence of Interleukin-2 receptor gamma proteinactivity; (b) the targeted genetic modification at the ApoE locusresults in a decrease in or absence of ApoE protein activity; (c) thetargeted genetic modification at the Rag1 locus results in a decrease inor absence of Rag1 protein activity; (d) the targeted geneticmodification at the Rag2 locus results in a decrease in or absence ofRag2 protein activity; or, (e) the targeted genetic modification at theRag2/Rag1 locus results in a decrease in or absence of Rag2 proteinactivity and i Rag1 protein activity.

56. The method of any one of embodiment 51-54, wherein the targetedgenetic modification of the Interleukin-2 receptor gamma locus comprises(a) a deletion of the entire rat Interleukin-2 receptor gamma codingregion or a portion thereof; (b) a replacement of the entire ratInterleukin-2 receptor gamma coding region or a portion thereof with ahuman Interleukin-2 receptor gamma coding region or a portion thereof;(c) a replacement of an ecto-domain of the rat Interleukin-2 receptorgamma coding region with the ecto-domain of a human Interleukin-2receptor gamma; or, (d) at least a 3 kb deletion of the Interleukin-2receptor gamma locus comprising the Interleukin-2 receptor gamma codingregion.

57. The method of any one of embodiment 51-55, wherein the targetedgenetic modification of the ApoE locus comprises: (a) a deletion of theentire ApoE coding region or a portion thereof; or, (b) at least a 1.8kb deletion of the ApoE locus comprising the ApoE coding region.

58. The method of any one of embodiment 51-55, wherein the targetedgenetic modification of the Rag2 locus comprises: (a) a deletion of theentire Rag2 coding region or a portion thereof; or, (b) at least a 5.7kb deletion of the Rag2 locus comprising the Rag2 coding region.

59. The method of any one of embodiment 51-55, wherein the targetedgenetic modification of the Rag1/Rag2 locus comprises: (a) a deletion ofthe entire Rag2 coding region or a portion thereof and a deletion of theentire Rag1 coding region or portion thereof; or, (b) a deletion of atleast 16 kb of the Rag2/Rag1 locus comprising the Rag2 and Rag1 codingregions.

60. The method of any one of embodiment 51-59, wherein the insertnucleic acid comprises an expression cassette comprising apolynucleotide encoding a selective marker.

61. The method embodiment 60, wherein the expression cassette comprisesa lacZ gene operably linked to an endogenous promoter at the genomiclocus and a human ubiquitin promoter operably linked to a selectivemarker gene.

62. The method of any one of embodiments 51-60, wherein the insertnucleic acid comprises a self-deleting selection cassette.

63. The method of embodiment 62, wherein the self-deleting selectioncassette comprises a selective marker operably linked to a promoteractive in the rat pluripotent cell and a polynucleotide encoding arecombinase operably linked to a male germ cell-specific promoter,wherein the self-deleting cassette is flanked by recombinationrecognition sites recognized by the recombinase.

64. The method of embodiment 63, wherein (a) the male germ cell-specificpromoter is a Protamine-1 promoter; or, (b) the recombinase gene encodesCre and the recombination recognition sites are loxP sites.

65. The method of embodiment 53, wherein the insertion of the exogenousnucleic acid sequence at the genomic locus comprises a reporter nucleicacid sequence operably linked to an endogenous Interleukin-2 receptorgamma promoter, an endogenous ApoE promoter, an endogenous Rag1promoter, or an endogenous Rag2 promoter.

66. The method of embodiment 65, wherein the reporter nucleic acidsequence encodes a reporter comprising β-galactosidase, mPlum, mCherry,tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus,YPet, enhanced yellow fluorescent protein (EYFP), Emerald, enhancedgreen fluorescent protein (EGFP), CyPet, cyan fluorescent protein (CFP),Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combinationthereof.

67. The method of any one of embodiment 51-66, wherein the pluripotentrat cell is a rat embryonic stem (ES) cell.

68. The method of any one of embodiment 51-67, wherein the pluripotentrat cell (a) is derived from a DA strain or an ACI strain; or, (b) ischaracterized by expression of a pluripotency marker comprising Oct-4,Sox-2, alkaline phosphatase, or a combination thereof; or, (c) ischaracterized by one or more of the following characteristics: (i) lackof expression of one or more pluripotency markers comprising c-Myc,Ecat1, and/or Rexo1; (ii) lack of expression of mesodermal markerscomprising Brachyury and/or Bmpr2; (iii) lack of expression of one ormore endodermal markers comprising Gata6, Sox17 and/or Sox7; or (iv)lack of expression of one or more neural markers comprising Nestinand/or Pax6.

69. The method of any one of embodiment 51-68, further comprisingidentifying the targeted genetic modification at the target genomiclocus, wherein the identification step employs a quantitative assay forassessing a modification of allele (MOA) at the target genomic locus.

70. The method of any one of embodiment 51-69, wherein introducing step(a) further comprises introducing a second nucleic acid encoding anuclease agent that promotes a homologous recombination between thetargeting vector and the target genomic locus in the pluripotent ratcell.

71. The method of embodiment 70, wherein the nuclease agent comprises achimeric protein comprising a zinc finger-based DNA binding domain fusedto a FokI endonuclease.

72. The method of embodiment 71, wherein the method results inbi-allelic modification of the target genomic locus.

73. The method of any one of embodiment 51-70, wherein introducing step(a) further comprises introducing into the pluripotent rat cell: (i) afirst expression construct comprising a first promoter operably linkedto a first nucleic acid sequence encoding a Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR)-associated (Cas) protein,(ii) a second expression construct comprising a second promoter operablylinked to a genomic target sequence linked to a guide RNA (gRNA),wherein the genomic target sequence is immediately flanked on the 3′ endby a Protospacer Adjacent Motif (PAM) sequence.

74. The method of embodiment 73, wherein the genomic locus of interestcomprises the nucleotide sequence of SEQ ID NO: 1.

75. The method of embodiment 73 or 74, wherein the gRNA comprises athird nucleic acid sequence encoding a Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) RNA (crRNA) and a trans-activatingCRISPR RNA (tracrRNA).

76. The method of embodiment 73, wherein the Cas protein is Cas9.

77. The method of embodiment 73, 74, or 75, wherein the gRNA comprises:(a) the chimeric RNA of the nucleic acid sequence of SEQ ID NO: 2; or,(b) the chimeric RNA of the nucleic acid sequence of SEQ ID NO: 3.

78. The method of embodiment 75, wherein the crRNA comprises SEQ ID NO:4; SEQ ID NO: 5; or SEQ ID NO: 6.

79. The method of embodiment 75, wherein the tracrRNA comprises SEQ IDNO: 7 or SEQ ID NO: 8.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1. Rat ES Cell Derivation and Characterization

1.1. Rat ES Cell Characterization

As shown in FIG. 1, rat ESCs grow as compact spherical colonies, whichroutinely detach and float in the dish (close-up, FIG. 7). Rat ESCsexpress pluripotency markers including Oct-4 (FIG. 2A) and Sox2 (FIG.2B), and express high levels of alkaline phosphatase (FIG. 3, leftpanel). Karyotype for line DA.2B is 42X,Y (FIG. 3, right panel). RatESCs often become tetraploid; thus, lines were pre-screened by countingmetaphase chromosome spreads; lines with mostly normal counts were thenformally karyotyped.

ACI blastocysts were collected from super-ovulated females obtainedcommercially. DA blastocysts were cultured from frozen 8-cell embryosobtained commercially. Zona pellucidae were removed with Acid Tyrodes;and blastocysts were plated onto mitotically inactivated MEFs.Outgrowths were picked and expanded using standard methods. Allblastocysts were plated, cultured and expanded using 2i media (Li et al.(2008) Germline competent embryonic stem cells derived from ratblastocysts, Cell 135:1299-1310; incorporated herein by reference in itsentirety).

TABLE 1 Rat ES Cell Derivation ACI DA Embryo source Blastocysts Frozen8-cell embryos (Superovulation) cultured to blastocyst Blastocystsplated: 107 22 Outgrowths: 32 (30% of blasts) 10 (45% of blasts) Lines:16 (50% of outgrowths)  9 (90% of outgrowths) Karyotyped: 3; all 42X, Y6: 3 42X, X  3 42X, Y GLT validated: 1 (ACI.G1) 1 42X, X (DA.2C) 1 42X,Y (DA.2B)

1.2. Rat Production

Chimeric rats were produced by blastocyst injection and transmission ofthe rat ESC genome. Chimeras produced by blastocyst microinjection usingparental ACI.G1 rat ESCs are shown in FIG. 8. F1 agouti pups with albinolittermates, sired by the ACI/SD chimera labeled with an asterisk (*) inFIG. 8 are shown in FIG. 9.

Germline Transmission of Parental Rat ESC.

Three euploid rat ESC lines were evaluated for pluripotency bymicroinjection into albino SD blastocysts. Chimeras were identified byagouti coat color, which indicates rat ESC contribution. For each line,a majority of chimeras transmitted the rESC genome to F1 offspring(Table 2).

TABLE 2 Germline Transmission of Parental rESC Germline Total pups rESC-GLT Chimeras trans- from GLT derived efficiency Line bred mitterschimeras pups (%) ACI.G1 5 3 (60%) 103 11 11 DA.2B 5 4 (80%) 129 11 9DA.2C (XX) 3 2 (66%) 45 7 16

1.3. Derivation of Rat Embryonic Stem Cells.

Superovulation protocol, rats

Day 0: injected with pregnant mare serum: IP, 20 U (0.4 ml).

Day 1: no action

Day 2: (46 hr. later): injected with hCG, IP, 50 U (1 ml).

-   -   set up single female matings.

Day 3: checked plugs. Females were plugged. This is day 0.5.

Day 6 (e3.5): Euthanized females and flushed embryos.

ES Cell Derivation Protocol (Superovulation)

Day 0:

-   -   1) Euthanized female rat with CO₂.    -   2) Swabbed ventral abdomen with 70% ethanol; using scissors,        opened the ventral body wall to expose the viscera.    -   3) Dissected out the oviducts and uterine horns and placed them        into a tissue culture dish containing warm N2B27 media. Washed        out as much blood as possible and transferred to a new dish with        N2B27.    -   4) Using a 1 ml syringe and a blunt 27 g needle, flushed media        through the uterine horns and oviducts to eject blastocysts into        the media.    -   5) Collected the blastocysts with a mouth pipet and transfer to        embryo culture dish containing KSOM+2i (1 μMPDW0325901, 3 μM        CHIR99021). KSOM is a culture medium produced by Millipore.        Catalog number is MR-106-D.    -   6) Cultured overnight at 37°; 7.5% CO₂.

ES Cell Derivation Protocol (Frozen Embryos)

Day 0:

-   -   1) Thawed frozen 8-cell embryos (commercially obtained) into M2        medium. Cultured 10 minutes at room temperature.    -   2) Transferred to KSOM+2i and culture overnight.

ES Cell Derivation Protocol (Same for Both)

Day 1:

-   -   1) Transferred cavitated embryos to 2i medium & culture        overnight.    -   2) Continued culturing un-cavitated embryos in KSOM+2i

Day 2:

-   -   1) Transferred all remaining embryos to 2i medium (whether or        not they've cavitated).    -   2) Cultured overnight; continued culturing earlier embryos in 2i        medium.

Day 3:

-   -   1) Transferred embryos for 30-60 seconds with Acid Tyrodes to        remove the zona pellucida.    -   2) Washed embryos 3× in 2i medium to remove Acid Tyrodes.    -   3) Deposited each embryo into a separate well of a 96-well        feeder plate (the well contains a monolayer of mitotically        inactivated mouse embryonic fibroblasts (MEFs).    -   4) Cultured overnight in 2i medium.

Day 4-5:

-   -   1) Monitored plated embryos for the presence of an outgrowth (an        amorphous undifferentiated mass of cells). Outgrowths are ready        for transfer when they are approximately twice the size of the        plated embryo.    -   2) Each day: remove spent media with a micropipette and replace        with fresh 2i media.    -   3) Transferred outgrowths to new feeder wells:        -   a. Removed spent media and gently wash well with PBS.        -   b. Removed PBS and add 30 μl 0.05% trypsin; incubate for 10            minutes.        -   c. Stopped trypsin reaction by adding 30 μl 2i+10% FBS.        -   d. Gently dissociated the cells with a micropipettor and            transferred entire contents of the well to a new well in a            24-well feeder plate. This was Passage 1 (P1).        -   e. Cultured overnight in 2i medium.

Day 5-8: (timing depends on how fast each line expands)

-   -   1) Changed media each day (2i media) and monitored for the        presence of colonies with an ESC morphology.    -   2) When colonies appear, continued culturing until colonies        expand to ˜50% confluency.

Ongoing:

-   -   1) Continued feeding and monitoring each line until        approximately 50% confluent.    -   2) Trypsinized cells as usual.    -   3) stopped trypsin with 2i+10% FBS; pelleted the cells by        centrifugation (5′, 1200 rpm in Beckman-Coulter tabletop        centrifuge).    -   4) Aspirated the supernatant and gently resuspend the cells in        400 μl Freezing Medium (70% 2i, 20% FBS, 10% DMSO).    -   5) Distributed the cells into 2 vials and freeze at −80°. This        was Passage 3 (P3).    -   6) For long-term storage, transferred the vials to liquid N2        storage.

The 2i media was prepared as follows in Table 3.

Reagent Vendor Concentration DMEM/F12 basal media Invitrogen/Life 1xTechnologies Neurobasal media Invitrogen/Life 1x TechnologiesPenicillin/streptomycin Invitrogen/Life  1% Technologies L-GlutamineInvitrogen/Life 4 mM Technologies 2-Mercaptoethanol Invitrogen/Life 0.1mM Technologies N2 supplement Invitrogen/Life 1x Technologies B27supplement Invitrogen/Life 1x Technologies LIF Millipore 100 U/mlPD0325901 (MEK Stemgent 1 uM inhibitor). CHIR99021 (GSK Stemgent 3 uMinhibitor).

Materials:

-   -   Pregnant Mare's Serum Gonadotropin (PMSG)    -   Human Pregnancy Urine Chorionic Gonadotropin (HCG)    -   Female Rats (5-12 weeks old)    -   Male rats (12 wks. to 8 mos. old), one per cage    -   Syringes/needles    -   Animal room with lights on 6:00-18:00

Procedure:

Day 1: 8:00-10:00 AM

-   -   Inject females with 20 IU PMSG (0.4 ml), IP    -   Discard unused PMSG.

Day 3: 8:00-10:00 AM (48 hours after PMSG injection)

-   -   Inject females with 50 IU HCG (1 ml), IP    -   Place one female per male in mating cage.    -   Discard unused HCG.

Day 4: 8:00-10:00 AM (24 hrs. after HCG injection)

-   -   Check females for plugs.

Hormone Suppliers

PMSG: Sigma #G-4877 (1000 IU). Resuspend in PBS to a final [ ] of 50IU/ml. Store at −20° in 1 ml aliquots.

HCG: Sigma #CG-5 (5000 IU). Resuspend in PBS to a final [ ] of 50 IU/ml.Store at −20° in 1 ml aliquots.

1.4.: Karyotyping of Rat Embryonic Stem Cell Lines

The rat ES cell lines generated herein were karyotyped, and the resultsare summarized in Tables 4-7.

TABLE 4 ACI.G1 Karyotyping Results Number of cells Number of cellskaryotyped 7 Number of cells analyzed 20 Number of 42, XY cells 18Number of abnormal cells 2 40, XY, −5, −9 1 41, XY, −14 1 42, XY 18Other notes: Two analyzed cells were missing different autosomes, whichmay be a sporadic occurrence due to technical artifact. 90% of analyzedcells had a normal male 42, XY karyotype. FIG. 4 provides a photographshowing the analysis of the chromosome number of the ACI.G1 rat ES cellline.

TABLE 5 DA.2B Karyotyping Results Number of cells Number of cellskaryotyped 6 Number of cells analyzed 20 Number of 42, XY cells 20Number of abnormal cells 0 42, XY 20 Other notes: All analyzed cells hada normal diploid 42, XY karyotype. FIG. 5 provides a photograph showingthe analysis of the chromosome number of the DA.2B rat ES cell line.

TABLE 6 DA.C2 Karyotyping Results Number of cells Number of cellskaryotyped 5 Number of cells analyzed 20 Number of 42, XY cells 20Number of abnormal cells 0 42, XX Other notes: 100% of analyzed cellshad normal female XX rat karyotype. FIG. 6 provides a photograph showingthe analysis of the chromosome number of the DA.C2 rat ES cell line.

TABLE 7 Blasto- Lines cysts Lines Karyo- strain plated established typedKaryotypes BN × SD 41 8 (20%) 5 all lines were high % F1 complexpolyploid ACI 27 16 (60%) 3 G1: 90% 42 XY; others were 70-85% euploid DA20 9 (45%) 6 2B: 100% 42 XY; 2C: 100% 42 XX; others were 95-100% euploidF344 4 1 (25%) 0 Totals 92 34 (37%)

1.5.: Electroporation of Vector into Rat Embryonic Stem Cell

1. Passaged rat ES cells 24-48 hrs prior to electroporation.

2. Changed media to RVG2i+ROCKi (10 μM Y-27632) 24 hr. prior toelectroporation

3. Changed media 30′ prior to trypsinization.

4. Aliquoted DNA to be electroporated.

5. Allowed DNA to warm at RT for >10 min.

6. Heated DNA for 5′ @ 62° C. Place DNA on ice.

7. Trypsinized cells:

-   -   a. Collected floating colonies. Washed plate to collect as many        floaters as possible.    -   b. Pelleted colonies: 3′ @ 750 rpm.    -   c. Washed pellet 1× with 5-10 ml PBS and re-spin/pellet    -   d. Aspirated supernatant; add 500λ trypsin, 0.05%+1% chicken        serum.        -   i. Did not pool more than 1 10 cm plate of colonies per            tube. If there are too many colonies packed into the bottom            of the tube during trypsinization they will clump and most            of the cells will be lost.    -   e. 4′ @ 37°. Pipetted colonies several times to minimize        clumping.    -   f. Repeated steps 1-2×: 4′ @ 37°.    -   g. Stopped trypsin with 500λ RVG2i+10% FBS.

8. Pelleted cells: 5′ @ 1200 rpm.

9. Resuspend cells in 10 ml PBS. Count two 20λ aliquots to determinetotal cell number.

10. Pelleted cells (5′/1200 rpm); calculate total cell number and totalresuspension volume to achieve correct cell concentration (target #175μl EP buffer).

11. Resuspend in a minimal volume of EP buffer; measure total volume andadjust to target volume with EP buffer. Electroporation buffer is soldby Millipore. The catalog # is ES-003-D. See, Valenzuela et al. (2003)Nature Biotechnology 21:652-659, which is herein incorporated byreference.

12. Add 75λ cells to 50λ DNA; transfer the 125λ cells/DNA solution toone well of a BTX 48-well cuvette.

-   -   a. Filled the empty wells in the same column with 125λ EP        buffer.

13. Pulsed the cuvette once in the BTX electroporator:

-   -   a. Settings: 400V; Ω; 100 μF (settings may vary)

14. Placed cuvette on ice for 15′ to recover.

15. Removed cells into 5 ml RVG2i+10 μM ROCKi.

16. Added to 15 cm plate with 20 ml RVG2i+1004 ROCKi. Plate has 2× neoRMEFs (or other MEFs depending on project). The neoR selectable marker isthe neomycin phosphotransferase (neo) gene of Beck et al. (1982) Gene,19:327-36 or in U.S. Pat. No. 7,205,148 or 6,596,541, each of which areherein incorporated by reference.

17. Incubated @ 37°. Begin selection 48 hrs later.

ROCK inhibitor used was Y-27632.

1.6: Selecting a Targeted Genetic Modification in a Rat Embryonic StemCell.

1. Passaged cells for 24-48 hrs prior to electroporation.

2. Changed media to RVG2i+ROCKi (1004 Y-27632) 24 hr. prior toelectroporation

3. Changed media 30′ prior to trypsinization.

4. Aliquoted DNA to be electroporated.

5. Allowed DNA warm at RT for >10 min.

6. Heated DNA for 5′ @ 62° C. Place DNA on ice.

7. Trypsinized cells:

-   -   a. Collected floating colonies. Washed plate to collect as many        floaters as possible.    -   b. Pelleted colonies: 3′ @ 750 rpm.    -   c. Washed pellet 1× with 5-10 ml PBS and re-spin/pellet    -   d. Aspirated supernatant; add 500λ trypsin, 0.05%+1% chicken        serum.        -   i. Did not pool more than 1 10 cm plate of colonies per            tube. If there are too many colonies packed into the bottom            of the tube during trypsinization they will clump and most            of the cells will be lost.    -   e. 4′ @ 37°. Pipetted colonies several times to minimize        clumping    -   f. Repeated 1-2×: 4′ @ 37°.

g. Stopped trypsin with 500λ RVG2i+10% FBS.

8. Pelleted cells: 5′ @ 1200 rpm.

9. Resuspended cells in 10 ml PBS. Count two 20λ aliquots to determinetotal cell number.

10. Pelleted cells (5′/1200 rpm); calculate total cell number and totalresuspension volume to achieve correct cell concentration (target #/75μl EP buffer).

11. Resuspend in a minimal volume of EP buffer; measured total volumeand adjusted to target volume with EP buffer.

12. Added 75λ cells to 50λ DNA; transfer the 125λ cells/DNA solution toone well of a BTX 48-well cuvette.

-   -   a. Filled the empty wells in the same column with 125λ EP        buffer.

13. Pulsed the cuvette once in the BTX electroporator:

-   -   a. Settings: 400V; 100 μF (settings may vary)

14. Placed cuvette on ice for 15′ to recover.

15. Removed cells into 5 ml RVG2i+10 μM ROCKi.

16. Added to 15 cm plate with 20 ml RVG2i+10 μM ROCKi. Plate had 2× neoRMEFs (or other MEFs depending on project).

17. Incubated @ 37°. Began selection 48 hrs later.

18. G418 selection protocol was as follows:

-   -   a. Day 2 (2^(nd) day after EP): incubated cells in 2i        media+G418, 75 μg/ml.    -   b. Day 3: incubated cells in 2i media without G418    -   c. Day 4: incubated cells in 2i media+G418, 75 μg/ml.    -   d. Day 5: incubated cells in 2i media without G418    -   e. Day 6: incubated cells in 2i media+G418, 75 μg/ml.    -   f. Day 7: incubated cells in 2i media without G418    -   g. Day 8: incubated cells in 2i media+G418, 75 μg/ml.    -   h. Day 9: incubated cells in 2i media without G418    -   i. Day 10: incubated cells in 2i media+G418, 75 μg/ml.    -   j. Day 11: incubated cells in 2i media without G418    -   k. Day 12: picked colonies to expand for screening. Each colony        was dissociated in 0.05% trypsin+1% chicken serum for 10 minutes        and then plated into 1 well of a 96-well feeder plate.

19. Expanded colonies for 3 days in 2i media.

20. Passaged clones 1:1 to new 96-well feeder plates.

21. Expanded clones for 3 days in 2i media.

22. For each clone, dissociated colonies in trypsin. Froze ⅔ of eachclone and store at −80°; plated the remaining ⅓ onto laminin plates(96-well plates coated with 10 μg/ml laminin).

23. When the laminin plates were confluent, passed off to the screeninglab for genotyping of the clones.

1.7. Molecular Signature of the Rat Embryonic Stem Cells

The genes listed in Table 8 were expressed at 20-fold lower in rat EScells than the corresponding genes in mouse ES cells. The genes listedin Table 9 were expressed at levels 20-fold higher in rat ES cells thanthe corresponding genes in mouse ES cells.

The microarray data in Tables 8 and 9 were generated as follows. Rat EScells (ACI.G2 and DA.2B) and mouse ES cells (F1H4) were cultured in 2imedia for 3 passages until confluent. F1H4 cells were cultured ongelatin-coated plates in the absence of feeders. F1H4 mouse ES cellswere derived from 12956/SvEvTac and C57BL/6NTac heterozygous embryos(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou, W. T., Auerbach, W.,Frendewey, D., Hickey, J. F., Escaravage, J. M., Esau, L., Dore, A. T.,Stevens, S., Adams, N.C., Dominguez, M. G., Gale, N. W., Yancopoulos, G.D., DeChiara, T. M., Valenzuela, D. M. (2007), incorporated by referenceherein in its entirety).

The following protocol was used for sample prep: The 1.5 mL Eppendorftubes were labeled with the Sample ID. Cells grown on a plate wererinsed in 37° C. Phosphate-Buffered Saline (PBS). PBS was removed and300 ul of Trizol® was added. A scraper was used to break the cells inTrizol® (Life Technology). The lysed cells were collected in Trizol® ina 1.5 mL Eppendorf tube. For cells grown on suspension, the cells wererinsed in 37° C. PBS and collected in a 1.5 mL tube. The cells were spundown; PBS was removed; and 300 ul of Trizol® was added to the cells. Thecell membranes were broken by pipetting. Samples were sorted for FACSwith 10 to 10⁵ cells, the volume was concentrated to less than 100 uL. 4volumes of RNA Lysis buffer were added and mixed by pipetting. Forsample, 320 uL RNA Lysis buffer was added to 80 uL sample. Samples werestored at −20° C.

RNA-Seq was used to measure the expression level of mouse and rat genes.Sequencing reads were mapped to mouse and rat reference genome byTophat, and RPKM (fragments per kilobase of exon per million fragmentsmapped) were calculated for mouse and rat genes. Homology genes based ongene symbol were selected, and then used t-test to compare theexpression level of each gene between mouse and rat. miR-632 was in thetop 10 highest expressed in rat ESCs but was not expressed in mouse EScells. Although no comparative data exist from miR-632, based on thelevel of its expression compared to other genes expressed in rat ESCsand their known function in embryonic development, miR-632 was selectedas a marker for rat ES cells.

TABLE 8 The genes listed were expressed at levels 20-fold lower in ratES cells than the corresponding genes in mouse ES cells. ID SymbolEntrez Gene Name Location Type(s) Abcb1b Abcb1b ATP-binding Plasmatransporter cassette, sub- Membrane family B (MDR/TAP), member 1B Acta2ACTA2 actin, alpha 2, Cytoplasm other smooth muscle, aorta Actg2 ACTG2actin, gamma 2, Cytoplasm other smooth muscle, enteric Aebp1 AEBP1 AEbinding protein 1 Nucleus peptidase Angptl2 ANGPTL2 angiopoietin-like 2Extracellular other Space Ankrd1 ANKRD1 ankyrin repeat Cytoplasmtranscription domain 1 (cardiac regulator muscle) Anxa1 ANXA1 annexin A1Plasma other Membrane Anxa6 ANXA6 annexin A6 Plasma other Membrane Anxa8ANXA8L2 annexin A8-like 2 Plasma other Membrane Arhgef25 ARHGEF25 Rhoguanine Cytoplasm other nucleotide exchange factor (GEF) 25 Axl AXL AXLreceptor Plasma kinase tyrosine kinase Membrane Basp1 BASP1 brainabundant, Nucleus transcription membrane attached regulator signalprotein 1 Bgn BGN biglycan Extracellular other Space Bst2 BST2 bonemarrow Plasma other stromal cell antigen 2 Membrane Btf3 BTF3 basictranscription Nucleus transcription factor 3 regulator Btg2 BTG2 BTGfamily, Nucleus transcription member 2 regulator Capsl CAPSLcalcyphosine-like Other other Cav1 CAV1 caveolin 1, Plasma transmembranecaveolae protein, Membrane receptor 22 kDa Ccdc80 CCDC80 coiled-coildomain Nucleus other containing 80 Ccnd2 CCND2 cyclin D2 Nucleus otherCd248 CD248 CD248 molecule, Plasma other endosialin Membrane Cd44 CD44CD44 molecule Plasma enzyme (Indian blood Membrane group) Cd97 CD97 CD97molecule Plasma G-protein Membrane coupled receptor Cdc42ep5 CDC42EP5CDC42 effector Cytoplasm other protein (Rho GTPase binding) 5 Cdh11CDH11 cadherin 11, type 2, Plasma other OB-cadherin Membrane(osteoblast) Cdkn2a CDKN2A cyclin-dependent Nucleus transcription kinaseinhibitor 2A regulator Cdo1 CDO1 cysteine Cytoplasm enzyme dioxygenasetype 1 Clip3 CLIP3 CAP-GLY domain Cytoplasm other containing linkerprotein 3 Cln5 CLN5 ceroid- Cytoplasm other lipofuscinosis, neuronal 5Cnn1 CNN1 calponin 1, basic, Cytoplasm other smooth muscle Col1a1 COL1A1collagen, type I, Extracellular other alpha 1 Space Col1a2 COL1A2collagen, type I, Extracellular other alpha 2 Space Col3a1 COL3A1collagen, type III, Extracellular other alpha 1 Space Col5a2 COL5A2collagen, type V, Extracellular other alpha 2 Space Col6a2 COL6A2collagen, type VI, Extracellular other alpha 2 Space Cryab CRYABcrystallin, alpha B Nucleus other Csf1 CSF1 colony stimulatingExtracellular cytokine factor 1 Space (macrophage) Cth CTH cystathionaseCytoplasm enzyme (cystathionine gamma-lyase) Cthrc1 CTHRC1 collagentriple Extracellular other helix repeat Space containing 1 Ctsc CTSCcathepsin C Cytoplasm peptidase Cyr61 CYR61 cysteine-rich, Extracellularother angiogenic inducer, Space 61 Ddx58 DDX58 DEAD (Asp-Glu- Cytoplasmenzyme Ala-Asp) box polypeptide 58 Dkk3 DKK3 dickkopf WNT Extracellularcytokine signaling pathway Space inhibitor 3 Dmc1 DMC1 DNA meioticNucleus enzyme recombinase 1 Dpysl3 DPYSL3 dihydropyrimidinase-Cytoplasm enzyme like 3 Dse DSE dermatan sulfate Cytoplasm enzymeepimerase Dusp1 DUSP1 dual specificity Nucleus phosphatase phosphatase 1Dusp27 DUSP27 dual specificity Other phosphatase phosphatase 27(putative) Dusp9 DUSP9 dual specificity Nucleus phosphatase phosphatase9 Ece2 ECE2 endothelin Plasma peptidase converting enzyme 2 MembraneEcm1 ECM1 extracellular matrix Extracellular transporter protein 1 SpaceEgr1 EGR1 early growth Nucleus transcription response 1 regulator Emp1EMP1 epithelial Plasma other membrane protein 1 Membrane Emp3 EMP3epithelial Plasma other membrane protein 3 Membrane Ephx2 EPHX2 epoxidehydrolase Cytoplasm enzyme 2, cytoplasmic F3 F3 coagulation factorPlasma transmembrane III (thromboplastin, Membrane receptor tissuefactor) Fau FAU Finkel-Biskis- Cytoplasm other Reilly murine sarcomavirus (FBR-MuSV) ubiquitously expressed Fbn1 FBN1 fibrillin 1Extracellular other Space Fbxo15 FBXO15 F-box protein 15 Othertranscription regulator Fhl2 FHL2 four and a half Nucleus transcriptionLIM domains 2 regulator Flnc FLNC filamin C, gamma Cytoplasm other FosFOS FBJ murine Nucleus transcription osteosarcoma viral regulatoroncogene homolog Fundc2 FUNDC2 FUN14 domain Cytoplasm other containing 2Gjb3 GJB3 gap junction Plasma transporter protein, beta 3, Membrane 31kDa Gpa33 GPA33 glycoprotein A33 Plasma other (transmembrane) MembraneGpbp1l1 GPBP1L1 GC-rich promoter Other other binding protein 1- like 1Gpc3 GPC3 glypican 3 Plasma other Membrane Grb10 GRB10 growth factorCytoplasm other receptor-bound protein 10 Gstm1 GSTM5 glutathione S-Cytoplasm enzyme transferase mu 5 Hap1 HAP1 huntingtin- Cytoplasm otherassociated protein 1 Hist1h2bc HIST2H2BE histone cluster 2, Nucleusother (includes H2be others) Hmga2 HMGA2 high mobility Nucleus enzymegroup AT-hook 2 Hmgn3 Hmgn3 high mobility Nucleus other groupnucleosomal binding domain 3 Hormad1 HORMAD1 HORMA domain Nucleus othercontaining 1 Hsd17b14 HSD17B14 hydroxysteroid Cytoplasm enzyme (17-beta)dehydrogenase 14 Hspb1 HSPB1 heat shock 27 kDa Cytoplasm other protein 1Hspb8 HSPB8 heat shock 22 kDa Cytoplasm kinase protein 8 Htra1 HTRA1HtrA serine Extracellular peptidase peptidase 1 Space Ifi204 Ifi204interferon activated Nucleus transcription (includes gene 204 regulatorothers) Ifi44 IFI44 interferon-induced Cytoplasm other protein 44 Ifit1IFIT1B interferon-induced Cytoplasm other protein with tetratricopeptiderepeats 1B Ifitm3 IFITM2 interferon induced Cytoplasm othertransmembrane protein 2 Igf2 IGF2 insulin-like growth Extracellulargrowth factor 2 Space factor (somatomedin A) Igfbp7 IGFBP7 insulin-likegrowth Extracellular transporter factor binding Space protein 7 Il1rl1IL1RL1 interleukin 1 Plasma transmembrane receptor-like 1 Membranereceptor Inhba INHBA inhibin, beta A Extracellular growth Space factorInhbb INHBB inhibin, beta B Extracellular growth Space factor Irf7 IRF7interferon Nucleus transcription regulatory factor 7 regulator Isg15ISG15 ISG15 ubiquitin- Extracellular other like modifier Space Itga5ITGA5 integrin, alpha 5 Plasma transmembrane (fibronectin Membranereceptor receptor, alpha polypeptide) Jun JUN jun proto-oncogene Nucleustranscription regulator Junb JUNB jun B proto- Nucleus transcriptiononcogene regulator Lgals3bp LGALS3BP lectin, galactoside- Plasmatransmembrane binding, soluble, 3 Membrane receptor binding proteinLgals9 LGALS9 lectin, galactoside- Extracellular other binding, soluble,9 Space Lmna LMNA lamin A/C Nucleus other Lox LOX lysyl oxidaseExtracellular enzyme Space Loxl2 LOXL2 lysyl oxidase-like 2Extracellular enzyme Space Loxl3 LOXL3 lysyl oxidase-like 3Extracellular enzyme Space Lrp1 LRP1 low density Plasma transmembranelipoprotein Membrane receptor receptor-related protein 1 Mageb16 MAGEB16melanoma antigen Other other family B, 16 Mcam MCAM melanoma cell Plasmaother adhesion molecule Membrane Mgp MGP matrix Gla proteinExtracellular other Space Mmp2 MMP2 matrix Extracellular peptidasemetallopeptidase 2 Space (gelatinase A, 72 kDa gelatinase, 72 kDa typeIV collagenase) Mxra8 MXRA8 matrix-remodelling Other other associated 8Myl9 MYL9 myosin, light chain Cytoplasm other 9, regulatory Mylpf MYLPFmyosin light chain, Cytoplasm other phosphorylatable, fast skeletalmuscle Nab2 NAB2 NGFI-A binding Nucleus transcription protein 2 (EGR1regulator binding protein 2) Ndufb4 NDUFB4 NADH Cytoplasm transporterdehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15 kDa Npm1 NPM1nucleophosmin Nucleus transcription (nucleolar regulator phosphoproteinB23, numatrin) Nr0b1 NR0B1 nuclear receptor Nucleus ligand- subfamily 0,group dependent B, member 1 nuclear receptor Nr4a1 NR4A1 nuclearreceptor Nucleus ligand- subfamily 4, group dependent A, member 1nuclear receptor Nrp2 NRP2 neuropilin 2 Plasma kinase Membrane Oas1aOAS1 2′-5′-oligoadenylate Cytoplasm enzyme synthetase 1, 40/46 kDa Oasl2Oasl2 2′-5′ oligoadenylate Other enzyme synthetase-like 2 P4ha2 P4HA2prolyl 4- Cytoplasm enzyme hydroxylase, alpha polypeptide II Parp3 PARP3poly (ADP-ribose) Nucleus enzyme polymerase family, member 3 PcolcePCOLCE procollagen C- Extracellular other endopeptidase Space enhancerPcyt1b PCYT1B phosphate Cytoplasm enzyme cytidylyltransferase 1,choline, beta Pdgfc PDGFC platelet derived Extracellular growth growthfactor C Space factor Phlda1 PHLDA1 pleckstrin Cytoplasm otherhomology-like domain, family A, member 1 Phlda2 PHLDA2 pleckstrinCytoplasm other homology-like domain, family A, member 2 Pla2g1b PLA2G1Bphospholipase A2, Extracellular enzyme group IB Space (pancreas) Pla2g4aPLA2G4A phospholipase A2, Cytoplasm enzyme group IVA (cytosolic,calcium- dependent) Porcn PORCN porcupine homolog Cytoplasm other(Drosophila) Postn POSTN periostin, Extracellular other osteoblastspecific Space factor Prrx1 PRRX1 paired related Nucleus transcriptionhomeobox 1 regulator Prss23 PRSS23 protease, serine, 23 Extracellularpeptidase Space Psmb8 PSMB8 proteasome Cytoplasm peptidase (prosome,macropain) subunit, beta type, 8 Ptgs2 PTGS2 prostaglandin- Cytoplasmenzyme endoperoxide synthase 2 (prostaglandin G/H synthase andcyclooxygenase) Ptn PTN pleiotrophin Extracellular growth Space factorPtrf PTRF polymerase I and Nucleus transcription transcript releaseregulator factor Rarg RARG retinoic acid Nucleus ligand- receptor, gammadependent nuclear receptor Rgs16 RGS16 regulator of G- Cytoplasm otherprotein signaling 16 Rn45s Rn45s 45S pre-ribosomal Other other RNARpl10a RPL10A ribosomal protein Other other L10a Rpl31 RPL31 ribosomalprotein Other other L31 Rpl37a RPL37A ribosomal protein Cytoplasm otherL37a Rps10 RPS10- RPS10-NUDT3 Cytoplasm other NUDT3 readthrough Rps14RPS14 ribosomal protein Cytoplasm translation S14 regulator Rps20 Rps20ribosomal protein Cytoplasm other S20 Rps26 RPS26 ribosomal proteinCytoplasm other S26 Rps9 RPS9 ribosomal protein Cytoplasm translation S9regulator S100a4 S100A4 S100 calcium Cytoplasm other binding protein A4S100a6 S100A6 S100 calcium Cytoplasm transporter binding protein A6Schip1 SCHIP1 schwannomin Cytoplasm other interacting protein 1 Sdc2SDC2 syndecan 2 Plasma other Membrane Serpine1 SERPINE1 serpin peptidaseExtracellular other inhibitor, clade E Space (nexin, plasminogenactivator inhibitor type 1), member 1 Serpine2 SERPINE2 serpin peptidaseExtracellular other inhibitor, clade E Space (nexin, plasminogenactivator inhibitor type 1), member 2 Serpinf1 SERPINF1 serpin peptidaseExtracellular other inhibitor, clade F Space (alpha-2 antiplasmin,pigment epithelium derived factor), member 1 Sh3gl2 SH3GL2 SH3-domainPlasma enzyme GRB2-like 2 Membrane Slc19a2 SLC19A2 solute carrier Plasmatransporter family 19 Membrane (thiamine transporter), member 2 Slc25a5SLC25A5 solute carrier Cytoplasm transporter family 25 (mitochondrialcarrier; adenine nucleotide translocator), member 5 Slc29a1 SLC29A1solute carrier Plasma transporter family 29 Membrane (equilibrativenucleoside transporter), member 1 Slc35f2 SLC35F2 solute carrier Otherother family 35, member F2 Snrpn SNRPN small nuclear Nucleus otherribonucleoprotein polypeptide N Snx22 SNX22 sorting nexin 22 Othertransporter Sparc SPARC secreted protein, Extracellular other acidic,cysteine- Space rich (osteonectin) Spp1 SPP1 secreted Extracellularcytokine phosphoprotein 1 Space Sult4a1 SULT4A1 sulfotransferaseCytoplasm enzyme family 4A, member 1 Tagln TAGLN transgelin Cytoplasmother Tcea3 TCEA3 transcription Nucleus transcription elongation factorA regulator (SII), 3 Tgfb3 TGFB3 transforming Extracellular growthgrowth factor, beta 3 Space factor Thbs1 THBS1 thrombospondin 1Extracellular other Space Thbs2 THBS2 thrombospondin 2 Extracellularother Space Tm4sf1 TM4SF1 transmembrane 4 L Plasma other six familymember 1 Membrane Tmbim1 TMBIM1 transmembrane Cytoplasm other BAXinhibitor motif containing 1 Tmem176b TMEM176B transmembrane Other otherprotein 176B Tnc TNC tenascin C Extracellular other Space Tpd52l1TPD52L1 tumor protein D52- Cytoplasm other like 1 Tpm2 TPM2 tropomyosin2 Cytoplasm other (beta) Usp18 USP18 ubiquitin specific Cytoplasmpeptidase peptidase 18 Vim VIM vimentin Cytoplasm other Wfdc2 WFDC2 WAPfour- Extracellular other disulfide core Space domain 2 Wisp2 WISP2 WNT1inducible Extracellular growth signaling pathway Space factor protein 2Ybx1 YBX1 Y box binding Nucleus transcription protein 1 regulator

TABLE 9 The genes listed were expressed at levels 20-fold higher in ratES cells than the corresponding genes in mouse ES cells. ID SymbolEntrez Gene Name Location Type(s) Ajap1 Ajap1 adherens junction Otherother associated protein 1 Amd1 AMD1 adenosylmethionine Cytoplasm enzymedecarboxylase 1 Ankrd2 ANKRD2 ankyrin repeat Nucleus transcriptiondomain 2 (stretch regulator responsive muscle) Arhgef9 ARHGEF9 Cdc42guanine Cytoplasm other nucleotide exchange factor (GEF) 9 Atp5h Atp5hATP synthase, H+ Cytoplasm enzyme transporting, mitochondrial F0complex, subunit d Btg3 BTG3 BTG family, Nucleus other member 3 Car6 CA6carbonic anhydrase Extracellular enzyme VI Space Camk4 CAMK4calcium/calmodulin- Nucleus kinase dependent protein kinase IV Capn12CAPN12 calpain 12 Other peptidase Cct6b CCT6B chaperonin Cytoplasmtransporter containing TCP1, subunit 6B (zeta 2) Cdx2 CDX2 caudal typeNucleus transcription homeobox 2 regulator Cldn5 CLDN5 claudin 5 Plasmaother Membrane Clec3a CLEC3A C-type lectin Other other domain family 3,member A Clic6 CLIC6 chloride intracellular Plasma ion channel channel 6Membrane Dhrsx DHRSX dehydrogenase/reductase Other enzyme (SDR family)X-linked Dpysl2 DPYSL2 dihydropyrimidinase- Cytoplasm enzyme like 2Dusp26 DUSP26 dual specificity Cytoplasm enzyme phosphatase 26(putative) Eci3 Eci3 enoyl-Coenzyme A Other enzyme delta isomerase 3Eef2k EEF2K eukaryotic Cytoplasm kinase elongation factor-2 kinase Efna1EFNA1 ephrin-A1 Plasma other Membrane Epha4 EPHA4 EPH receptor A4 Plasmakinase Membrane Fank1 FANK1 fibronectin type III Nucleus transcriptionand ankyrin repeat regulator domains 1 Fhit FHIT fragile histidineCytoplasm enzyme triad Filip1 FILIP1 filamin A Cytoplasm otherinteracting protein 1 Fmod FMOD fibromodulin Extracellular other SpaceFoxe1 FOXE1 forkhead box E1 Nucleus transcription (thyroid regulatortranscription factor 2) Fry FRY furry homolog Extracellular other(Drosophila) Space Gjb5 GJB5 gap junction protein, Plasma transporterbeta 5, 31.1 kDa Membrane Gpx2 GPX2 glutathione Cytoplasm enzymeperoxidase 2 (gastrointestinal) Grxcr2 GRXCR2 glutaredoxin, Other othercysteine rich 2 Hecw2 HECW2 HECT, C2 and WW Extracellular enzyme domaincontaining Space E3 ubiquitin protein ligase 2 Hey2 HEY2hairy/enhancer-of- Nucleus transcription split related with regulatorYRPW motif 2 Icos Icos inducible T-cell co- Plasma other stimulatorMembrane Ifitm1 IFITM1 interferon induced Plasma transmembranetransmembrane Membrane receptor protein 1 Il1f8 IL1F8 Interleukin-1Extracellular cytokine (IL36B) family member space (Interleukin 36 beta)Il28ra IL-28RA Interleukin 28 receptor, Plasma Cytokine alpha membranereceptor Igfbpl1 IGFBPL1 insulin-like growth Other other factor bindingprotein-like 1 Ipcef1 IPCEF1 interaction protein Cytoplasm enzyme forcytohesin exchange factors 1 Lctl Lctl lactase-like Cytoplasm other LdhdLDHD lactate Cytoplasm enzyme dehydrogenase D Lef1 LEF1 lymphoidenhancer- Nucleus transcription binding factor 1 regulator Lefty1 LEFTY1left-right Extracellular growth factor determination factor 1 Space LifrLIFR leukemia inhibitory Plasma transmembrane factor receptor alphaMembrane receptor Lpar2 LPAR2 lysophosphatidic Plasma G-protein acidreceptor 2 Membrane coupled receptor Mog MOG myelin Extracellular otheroligodendrocyte Space glycoprotein Morn5 MORN5 MORN repeat Other othercontaining 5 Pigz NCBP2 nuclear cap binding Nucleus other proteinsubunit 2, 20 kDa Nptxr NPTXR neuronal pentraxin Plasma transmembranereceptor Membrane receptor Ntm NTM neurotrimin Plasma other MembraneNutf2 NUTF2 nuclear transport Nucleus transporter factor 2 Ocln OCLNoccludin Plasma enzyme Membrane Olr1 OLR1 oxidized low density Plasmatransmembrane lipoprotein (lectin- Membrane receptor like) receptor 1Pabpc4 PABPC4 poly(A) binding Cytoplasm translation protein, cytoplasmicregulator 4 (inducible form) Pde11a PDE11A phosphodiesterase Cytoplasmenzyme 11A Pdyn PDYN prodynorphin Extracellular transporter Space Per3PER3 period circadian Nucleus other clock 3 Pllp PLLP plasmolipin Plasmatransporter Membrane Ppp1r14c PPP1R14C protein phosphatase Cytoplasmother 1, regulatory (inhibitor) subunit 14C Pramel6 Pramel6preferentially Other other expressed antigen in melanoma like 6 Ptpn18PTPN18 protein tyrosine Nucleus phosphatase phosphatase, non- receptortype 18 (brain-derived) Pycr1 PYCR1 pyrroline-5- Cytoplasm enzymecarboxylate reductase 1 Rab26 RAB26 RAB26, member Plasma enzyme RASoncogene Membrane family Ramp2 RAMP2 receptor (G protein- Plasmatransporter coupled) activity Membrane modifying protein 2 Rbm24 RBM24RNA binding motif Other other protein 24 Rhag RHAG Rh-associated Plasmapeptidase glycoprotein Membrane Rpl3 RPL3 ribosomal protein Cytoplasmother L3 Sall3 SALL3 sal-like 3 Nucleus other (Drosophila) Satb1 SATB1SATB homeobox 1 Nucleus transcription regulator Scg2 SCG2 secretograninII Extracellular cytokine Space Slc15a1 SLC15A1 solute carrier familyPlasma transporter 15 (oligopeptide Membrane transporter), member 1Slc1a1 SLC1A1 solute carrier family 1 Plasma transporter(neuronal/epithelial Membrane high affinity glutamate transporter,system Xag), member 1 Slc24a5 Slc24a5 solute carrier family Other other24 (sodium/potassium/calcium exchanger), member 5 Slc37a2 SLC37A2 solutecarrier family Other transporter 37 (glucose-6- phosphate transporter),member 2 40424 SNTB1 syntrophin, beta 1 Plasma other (dystrophin-Membrane associated protein A1, 59 kDa, basic component 1) St6galnac3ST6GALNAC3 ST6 (alpha-N- Cytoplasm enzyme acetyl-neuraminyl-2,3-beta-galactosyl- 1,3)-N- acetylgalactosaminide alpha-2,6-sialyltransferase 3 Tex12 TEX12 testis expressed 12 Nucleus other Tex15TEX15 testis expressed 15 Extracellular other Space Tfap2a TFAP2Atranscription factor Nucleus transcription AP-2 alpha regulator(activating enhancer binding protein 2 alpha) Tmc1 TMC1 transmembranePlasma other channel-like 1 Membrane Tmem130 TMEM130 transmembrane Otherother protein 130 Tmem30b TMEM30B transmembrane Other other protein 30BTomm20 TOMM20 translocase of outer Cytoplasm transporter mitochondrialmembrane 20 homolog (yeast) Tox3 TOX3 TOX high mobility Other othergroup box family member 3 Ttc25 TTC25 tetratricopeptide Cytoplasm otherrepeat domain 25 Tymp TYMP thymidine Extracellular growth factorphosphorylase Space Ubb Ubb ubiquitin B Cytoplasm other Vamp7 VAMP7vesicle-associated Cytoplasm transporter membrane protein 7 Wfdc12Wfdc12 WAP four-disulfide Extracellular other core domain 12 SpaceWfdc15a Wfdc15a WAP four-disulfide Other other core domain 15A Wfdc6aWfdc6a WAP four-disulfide Other other core domain 6A

TABLE 10 A subset of genes from Table 9, which are expressed at levels20-fold higher in rat ES cells than the corresponding genes in mouse EScells. ID Entrez Gene Name Ajap1 Adheres Junctions Associate ProteinCldn5 Claudin 5 Arhgef9 Cdc42 guanine nucleotide exchange facter 9 Camk4Calcium/calmodulin-dependent protein kinase IV Efna1 ephrin-A1 Epha4 EPHreceptor A4 Gjb5 gap junction protein beta 5 Igfbpl1 Insulin-like growthfactor binding protein-like 1 Il1f8 Interleukin 36 beta Il28raInterleukin 28 receptor, alpha Lefty1 left-right determination factor 1Lifr Leukemia inhibitory factor receptor alpha Lpar2 Lysophosphatidicacid receptor 2 Ntm Neuronal pentraxin receptor Ptpn18 Protein tyrosinephosphatase non-receptor type 18 Cdx2 Caudal type homeobox 2 Fank1Fibronectin type III and ankyrin repeat domains 1 Foxe1 Forkhead box E1(thyroid transcription factor 2) Hey2 Hairy/enhancer-of-split relatedwith YRPW motif 2 Lef1 Lymphoid enhancer-binding factor 1 Sall3 Sal-like3 (Drosophila) Satb1 SATB homeobox 1

An additional molecular signature employing the pluripotencymarkers/genes for the rat ES cells has also been developed. Table 11provides a gene list and their expression ranks from the RNA profilingdata. mRNA was isolated from rat ES cells and the expression level ofvarious markers were compared relative to each other. The term “rank”means the comparative expression levels of individual genes: the higherthe rank (1 is highest), the higher the expression. For example, Oct4'srank of 13 means that, of all the genes assayed, it was expressed higherthan all but 12 genes. Background in this experiment was any expressionvalue below 30; 6107 genes had expression values of 30 or higher.

TABLE 11 Rat ES cell molecular signature employing various pluripotency,mesodermal, endodermal, neural and trophectoderm markers/genes.Pluripotency Mesodermal Endodermal Neural Trophectoderm PluripotencyRank Mesodermal Rank Endodermal Rank Neural Rank Trophectoderm Rankc-Myc 8248 Brachyury 7542 Gata6 11195 Nestin 7761 Cdx2 739 Dnmt3L 127Flk1 Not Sox17 11418 Pax6 13570 tested Dppa2 Not tested Nodal 3050 Hhex14571 Sox2 681 Dppa5 Not tested Bmp4 3072 Nodal 3050 Ecat1 9714 Bmpr26382 Ext1 6091 Eras 2541 Sox7 10284 Err-beta 1368 Fbxo15 1369 Fgf4 3440Fthl17 Not tested Gdf3 2771 Rank >6107 = bkg expression Klf4 836 Lef11313 LIF receptor 724 Lin28 828 Nanog 774 Oct4 13 Rexo1 6119 Sox15 4524Sox2 681 SSEA1 Not tested SSEA4 Not tested Stella Not tested Tcl1 Nottested Utf1 1501

Example 2: Inactivation of Genomic Loci in Rats

2.1: Inactivation of Endogenous Genomic Loci Using an Endonuclease Agent

In order to introduce a mutant allele at an endogenous rat genomiclocus, the rat ES cells described herein are electroporated withexpression vectors (or mRNA) that express ZFNs 1 and 2 (or TALENs 1 and2). These proteins bind their target sequences on opposite strands,separated by about 6 bp to about 40 bp. A double-stranded break isformed within the target locus, which the cell attempts to repair byNon-Homologous End-Joining (NHEJ). In many cases, NHEJ results increation of a deletion, which often disrupts the function of the gene(most often by producing a frameshift mutation). In order to identify apositive clone comprising a mutant allele, the electroporated cells areplated at low density, because no drug selection is done. Colonies arepicked and assayed at the target site to see if a mutation was produced(e.g., using a modification of allele (MOA) assay described above). Theselected ES cells comprising the mutant allele are then introduced intoa host rat embryo, for example, a pre-morula stage or blastocyst stagerat embryo, and implanted in the uterus of a surrogate mother togenerate a founder rat (F0 rat). Subsequently, the founder rat is bredto a wild-type rat to create F1 progeny heterozygous for the mutantallele. Mating of the heterozygous F1 rat can produce progeny homozygousfor the mutant allele.

2.2.: Rat ESC Targeting for the Inactivation of the Rat Apolipoprotein E(ApoE) Gene Using Zinc Finger Nucleases

Zinc finger nucleases use sequence specific modular DNA binding domainsto direct endonuclease activity to unique target sequence in the genome.ZFNs are engineered as a pair of monomers. Each monomer containsnonspecific cleavage domain from FokI endonuclease fused to 3 or morezinc finger DNA-binding domains. Each zinc finger binds a 3 bp subsiteand specificity is achieved by the combined target sites of bothmonomers. ZFNs produce double-stranded breaks (DSB'S) in DNA, andmutations (insertions or deletions) frequently occur duringnon-homologous end joining (NHEJ). DSBs also stimulate homology-directedrepair (HDR) by homologous recombination if a donor sequence is providedwith ZFN.

Such ZFNs were employed in combination with the various methods andcompositions described herein to improve targeting efficiency. The ratApolipoprotein E (ApoE) locus was targeted as described in Example3.2(a)(i), except expression vectors that express ZFNs 1 and 2 were alsointroduced into the rat ES cells. See FIG. 10 which provides a schematicof the ApoE targeting event in combination with rTZFN1P and rTZFN2P. Thetargeting efficiency was determined as discussed below in Example 6 andresults are shown in FIG. 11. Surprisingly, the targeting efficiencywent up 8-10 fold.

A plasmid targeting vector was built with a self-deleting drug selectioncassette and a lacZ gene as a reporter gene. Good targeting efficiencywas achieved and a high % chimeras were produced. Zinc finger nucleases(ZFNs) were also tested in combination with targeting vectors to examineits effect on improving targeting efficiency. The targeting vector wasco-expressed with the expression vectors for 2 ZFN pairs that cut theApoE locus. The rat ESC clones electroporated with both the targetingvector and a set of the ZFNs showed a targeting efficiency of 8-10 foldhigher than that of rat ESC clones electroporated with a targetingvector alone. Moreover, bi-allelic homozygous targeting in about 2% ofour clones was detected. High % chimeras from two of these targetedclones were obtained.

The ApoE-targeted (with ZFN assistance) rat ESC clones weremicroinjected into SD blastocysts, which were then transferred topseudopregnant SD recipient females, using standard techniques. Chimeraswere identified by coat color; male F0 chimeras were bred to SD females.Germline F1 pups were genotyped for the presence of the targeted ApoEallele (FIG. 17). There was a high % chimeras from two of these targetedclones.

An ApoE knockout rat provides a means to study various types ofdisorders and diseases. In humans, Apolipoprotein is found inchylomicron, HDL, LDL and VLDL. ApoE is essential for the normalcatabolism of triglyceride-rich lipoprotein constituents. Defects inAPOE result in numerous disease states including, for example, familialhypercholesterolemia, hyperlipemia, betalipoproteinemia, familialdysbetalipoproteinemia, type III hyperlipoproteinemia (HLP III), risk ofcoronary artery disease. One isoform (ApoE4) is associated withlate-onset familial and sporadic Alzheimer's disease, possibly with MSas well.

In mice, ApoE is primarily found in HDL; transports cholesterol, as inhumans. ApoE-deficient mice (2 independent KOs) have 5 times normalplasma cholesterol; developed foam cell-rich depositions in theirproximal aortas by age 3 months (comparable to human syndrome).

ApoE knockouts in rats offer an animal model to study endothelialfunction, including, but not limited to, plaque formation,transcriptional changes (RNA-Seq), ex vivo function. Moreover, largersize of rats would facilitate all these assays and potentially improvethe quality of the RNA-Seq data.

2.3. Inactivation of the Rat Interleukin-2 Receptor Gamma (IL2r-γ) LocusUsing Zinc Finger Nucleases

The rat Interleukin-2 receptor gamma (IL2r-γ) locus was targeted asdescribed in Example 3.3(a), except that expression vectors that expressZFN U (ZFN upstream) and ZFN D (ZFN downstream) were also introducedinto the rat ES cells. FIG. 18 provides a schematic of the IL2r-γtargeting event in combination with ZFN U and ZFN D. The sequence of theIL2r-γ locus which these zinc fingers bind is denoted in FIG. 18. Thetargeting efficiency was determined as discussed below in Example 3.3(a)and the results are shown in FIG. 18. Briefly, homozygously targetedclones were confirmed by PCR. For the ZFN1 pair: 173 mutant clones outof 192 screened (90%) and for the ZFN2 pair: 162 clones out of 192 (84%)screened.

The IL2r-γ-targeted (with ZFN assistance) rat ESC clones weremicroinjected into SD blastocysts, which were then transferred topseudopregnant SD recipient females, using standard techniques. Chimeraswere identified by coat color; male F0 chimeras were bred to SD females.Germline F1 pups were genotyped for the presence of the targeted IL2r-γallele.

2.4.: Inactivation of the Rat Interleukin-2 Receptor Gamma (IL2r-γ)Using CRISPR/Cas9

The rat IL2r-γ locus was targeted as described in Example 3.3(a), exceptthat the CRISPR/Cas9 system was also introduced into the rat ES cells toaid in targeting efficiency. SBI: System Biosciences Cas9“SmartNuclease” all-in-one vectors were employed and Cas9 expression wasdriven by CAG, EF1a, PGK, or CMV promoter. Custom gRNA was ligated intoa vector and expressed by H1 promoter. 4 gRNAs against Il2rg weredesigned. The targeting efficiency when employing the various guide RNAsis shown in FIG. 19.

Example 3: Targeted Modification of Rat Genomic Loci

3.1: Rat ESC Targeting: The Rat Rosa 26 Locus.

The rat Rosa26 locus lies between the Setd5 and Thumpd3 genes as inmouse, with the same spacing. The rat Rosa 26 locus (FIG. 12, Panel B)differs from the mouse Rosa 26 locus (FIG. 12, Panel A). The mouseRosa26 transcripts consist of 2 or 3 exons. The rat locus contains a 2ndexon 1 (Ex1b) in addition to the homologous exon to mouse exon1 (Ex1a).No 3rd exon has been identified in rat. Targeting of a rat Rosa26 alleleis depicted in FIG. 12 (bottom), where homology arms of 5 kb each werecloned by PCR using genomic DNA from DA rat ESC. The targeted allelecontains a SA (splicing acceptor)-lacZ-hUb-neo cassette replacing a 117bp deletion in the rat Rosa26 intron.

Targeting efficiency at the rat Rosa 26 locus was determined (Table 12).Linearized vector was electroporated into DA or ACI rat ESCs, andtransfected colonies were cultured in 2i media+G418, using standardtechniques. Individual colonies were picked and screened using a Loss ofAllele (LOA) assay (Valenzuela, D. et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech. 21:652-660, incorporated herein by reference).

TABLE 12 rat Rosa26 Targeting Efficiency Colonies Reconfirmed TargetingCell line picked positives efficiency (%) DA.2B 192 4 2.1 ACI.G1 96 44.2

Chimera Production and Germline Transmission Using Rosa26-Targeted RatESC Clones.

Reconfirmed Rosa26-targeted rat ESC clones were microinjected into SDblastocysts, which were then transferred to pseudopregnant SD recipientfemales, using standard techniques. Chimeras were identified by coatcolor; male F0 chimeras were bred to SD females. Germline (agouti) F1pups were genotyped for the presence of the targeted Rosa26 allele; nineof 22 agouti pups genotyped as heterozygous at the Rosa26 locus (Table13).

TABLE 13 Germline Transmission Using Targeted Rosa26 rESC ESC- R26Clones Germline rESC- derived Cell clones producing Transmitting Totalderived pups line injected Chimeras Clones Pups Pups (%) DA.2B 4 3 2AH7: 64 AH7: 41 AH7: 63 AE3: 112 AE3: 6 AE3: 3 ACI.G1 4 4 1 DE9: 39 DE9:4 10

3.2. (a)(i): Targeting of the Rat Apolipoprotein E (ApoE) Locus.

The rat Apolipoprotein E (ApoE) locus was targeted to disrupt ApoEfunction. Targeting of the ApoE locus was done using a targeting vectorcomprising a lacZ-hUb-neo cassette flanked with a 5′ and 3′ homologyarms homologous to the ApoE locus. FIG. 20 depicts a geneticallymodified rat ApoE locus that has been disrupted by a 1.8 kb deletion andthe insertion of a lacZ-hUb-neo cassette, which further includes aself-deleting Cre cassette comprising a Crei gene driven by a protaminepromoter. The electroporation conditions were as follows: 6 ug DNA;2.05×10⁶ cells; 400V; 200 uF: 342 V, 593 usec; plate on 15 cm 2× denseneoR MEFs in 2i+10 uM ROCKi.

Targeting efficiency at the ApoE locus was determined and is shown inTable 14. Linearized vector was electroporated into DA.2B rat ESCsderived from the DA strain, and transfected colonies were cultured usingstandard techniques. Individual colonies were picked and screened usinga Loss of Allele (LOA) assay.

TABLE 14 rat ApoE Targeting Efficiency Colonies Targeting Cell lineVector picked Targeted efficiency (%) DA.2B ApoE-mSDC 192 7 3.7 DA.2BApoE-mSDC 192 15 7.8

Chimera production and germline transmission using ApoE-targeted rat ESCclones was performed. ApoE-targeted rat ESC clones were microinjectedinto SD blastocysts, which were then transferred to pseudopregnant SDrecipient females, using standard techniques. Chimeras were identifiedby coat color; male F0 chimeras were bred to SD females. F1 pups weregenotyped for the presence of the targeted ApoE allele (Table 15).

TABLE 15 Microinjection Results Exp Clone pups Chimeras 1 ApoE-AF5 4 3(90, 90, 90) 2 ApoE-BC4 5 0

Additional targeting data for ApoE is also provided in FIG. 21.

3.2.(a)(ii). Targeting ApoE in Rats with a Targeting Vector

FIG. 20 provides a schematic of the rat ApoE locus and a targetingplasmid. The upper schematic of FIG. 20 shows the genomic structure ofthe rat ApoE locus and the genomic regions corresponding to 5′ and 3′homology arms (5 kb and 5.4 kb, respectively; dark grey boxes). Exon 1of ApoE is non-coding and is shown as an open box closest to the 5′homology arm. The 3 introns of ApoE are denoted as lines and exons 2 and3 comprise coding regions and are shown as stippled grey boxes. Exon 4contains both coding and non-coding sequences as denoted by the stippledgrey shading and the open box.

The lower schematic in FIG. 20 is the targeting vector. The 5′ and 3′homology arms (5 kb and 5.4 kb respectively) are denoted by the darkgrey boxes. The targeting vector comprises a reporter gene (lacZ) and aself-deleting cassette flanked by loxP sites (open arrows). Theself-deleting cassette comprises the Crei gene operably linked to amouse Prm1 promoter and a selection cassette comprising a neomycinresistance gene operably linked to a human ubiquitin promoter.

The Crei gene comprises two exons encoding a Cre recombinase, which areseparated by an intron (Crei) to prevent its expression in a prokaryoticcell. See, See, for example, U.S. Pat. No. 8,697,851 and U S ApplicationPublication 2013-0312129, which describe the self-deleting cassette indetail and are hereby incorporated by reference in their entirety. Byemploying the Prm1 promoter, the self-deleting cassette can be deletedspecifically in male germ cells of F0 rats. The targeting vector waselectroporated into the rat ES cells obtained in Example 1 and the cellswere plated on 15 cm 2× dense neomycin-resistant MEFs in 2i+10 uM ROCKi.The transformed rat ES cells were cultured, selected, and maintained asdescribed in Example 1.

As shown in Table 23, 384 colonies were screened and 23 targeted cloneswere obtained. The targeting efficiency was 5.99%. 3 clones wereinjected into blastocysts as described herein in Example 1. 3 clonesproducing chimeras were obtained and 2 of the clones transmitted thetargeted modification through the germline.

3.2.(a)(iii). Targeting ApoE in Rats with a Targeting Vector inCombination with Zinc Finger Nucleases

The targeting vector employed in Example 3.2(a)(ii) was used incombination with zinc finger nucleases to target the rat ApoE locus.Table 16 provides a summary of the genomic organization of the rat ApoElocus. The positions shown in the Table 16 were taken from build 5.0 ofthe Reference Sequence of the rat genome (ENSMBL). ApoE is on chromosome1 on the (−) strand.

TABLE 16 Summary of the rat ApoE locus and the positions of the zincfinger nuclease binding sites and cutting sites. Feature Start Endlength Notes Exon 1 81881110 81881182   73 5′ non-coding Exon2 8188026981880332   64 contains ATG ATG 81880309 81880311    3 start codon Exon381879607 81879775  169 ZFN1a binding site 81879707 81879693   15CAGGCCCTGAACCGC (SEQ ID NO: 10) ZFN1 cutting site 81879692 81879687    6TTCTGG (SEQ ID NO: 11) ZFNlb binding site 81879686 81879671   16GATTACCTGCGCTGGG (SEQ ID NO: 12) Intron 3-4 81879776 81879207  400ZF21a binding site 81879591 81879577   15 TTCACCCTCCGCACC (SEQ IDNO: 13) ZFN2 cutting site 81879576 81879570    7 TGCTGAG (SEQ ID NO: 14)ZF21b binding site 81879569 81879552   18 TATCCAGATCCAGGGGTT(SEQ ID NO: 15) Exon 4 81878371 81879208  838 contains TGA TGA 8187848281878484    3 ApoE deletion 81878482 81880311 1830

FIG. 10 provides a schematic of the rat ApoE locus and denotes with greybars the cutting site for ZFN1 and ZFN2. The cutting site for ZFN1 is inexon 3 and the cutting site for ZNF2 is in intron 3. The exact positionof the both ZFN sites is set forth in Table 16. The genomic regionscorresponding to the 5′ and 3′ homology arms (5 kb and 5.4 kb,respectively) are denoted by the dark grey boxes. Exon 1 of ApoE isnon-coding and is shown as an open box closest to the 5′ homology arm.The three introns of the ApoE gene are denoted as lines and exons 2 and3 comprise coding regions and are shown as stippled grey boxes. Exon 4contains both coding and non-coding sequences as denoted by the stippledgrey shading and the open box.

The employed targeting vector was the same as that in Example 3.2(a)(ii)and shown in FIG. 20. The ZFNs were introduced as two expressionplasmids, one for each half of the ZFN pair. 20 ug of the plasmid forZFN1 and 20 ug of the plasmid for ZFN2 was used. ZFNs were purchasedfrom Sigma. The expression of each ZFN was driven by the CMV promoter.

The targeting vector were electroporated into the rat ES cells obtainedin Example 1 and the cells were plated on 15 cm 2× dense neoR MEFs in2i+10 uM ROCKi. The transformed rat ES cells were cultured, selected andmaintained as described in Example 1.

As shown in Table 23, 384 colonies were screened and 290 targeted cloneswere obtained. The targeting efficiency was 75.52%. 2 clones wereinjected into blastocysts as described herein in Example 1. Two clonesproducing chimeras were obtained and one of the clones transmitted thetargeted modification through the germline.

Moreover, employing ZFN1 and ZFN2 produced 8 biallelic targeted cloneswith an efficiency of 2.08%.

3.2. (b)(i): Targeted Modification of the Rat Apolipoprotein E (ApoE)Locus Using a Large Targeting Vector (LTC).

Targeting of the ApoE locus is done using a large targeting vector(LTVEC) comprising a lacZ-mouse Prm1-Crei cassette flanked with a 5′homology arm to the ApoE locus of about 45 kb and a 3′ homology arm tothe ApoE locus of about 23 Kb. FIG. 22 depicts the rat ApoE locus inwhich the ApoE locus has been disrupted by a 1.83 kb deletion and theinsertion of the lacZ gene and a self-deleting cassette comprisingmPrm1-Crei cassette and a hUb-neo selection cassette. Methods employedin example 3.2(a)(i) can be used to introduce this vector into rat EScells.

Example 3.2. (b)(ii). Targeting of the Rat ApoE Locus with a LargeTargeting Vector (LTVEC)

FIG. 22 provides a schematic of the rat ApoE locus and a large targetingvector (LTVEC). The upper schematic of FIG. 22 shows the genomicorganization of the rat ApoE locus and the genomic regions correspondingto the 5′ and 3′ homology arms (45 kb and 23 kb, respectively; dark greyboxes). Exon 1 of ApoE is non-coding and is shown as an open box closestto the 5′ homology arm. The 3 introns of ApoE are denoted as lines andexons 2 and 3 comprise coding regions and are shown as stippled greyboxes. Exon 4 contains both coding and non-coding sequences as denotedby the stippled grey shading and the open box.

The lower schematic in FIG. 22 is the LTVEC. The 5′ and 3′ homology arms(45 kb and 23 kb, respectively) are denoted by the dark grey boxes. Thetargeting vector comprises a reporter gene (lacZ) and a self-deletingcassette flanked by loxP sites (open arrows), which comprises the Creigene operably linked to a mouse Prm1 promoter and a drug selectioncassette comprising a neomycin resistance gene operably linked to ahuman ubiquitin promoter. The Crei comprises two exons encoding the Crerecombinase which are separated by an intron (Crei) to prevent itsexpression in a prokaryotic cell. See, for example, U.S. Pat. No.8,697,851 and U.S. Application Publication 2013-0312129, which describesthe self-deleting cassette in detail and is hereby incorporated byreference in their entirety. By employing a mouse Prm1 promoter, theself-deleting cassette can be deleted specifically in male germ cells ofF0 rat.

The LTVEC was electroporated into the rat ES cells obtained in Example 1and the cells were plated on 15 cm 2× dense neoR MEFs in 2i+10 uM ROCKi.The transformed rat ES cells were cultured, selected, and maintained asdescribed in Example 1.

As shown in Table 23, 288 colonies were screened and 8 targeted cloneswere obtained. The targeting efficiency was 2.78%. 3 clones wereinjected into a host embryo at a blastocyst stage as described herein inExample 2 to produce chimeric rats (F0). Moreover, one biallelictargeted clone was produced providing a biallelic efficiency of 0.35%.

3.2. (b)(iii). Targeting ApoE in Rats with a Large Targeting Vector(LTVEC) in Combination with Zinc Finger Nucleases

The LTVEC employed in Example 3.2.(b)(ii) was used in combination withzinc finger nucleases to target the rat ApoE locus. Table 16 provides asummary of the genomic organization of the rat ApoE locus and thepositions shown were taken from build 5.0 of the Reference Sequence ofthe rat genome (ENSMBL).

FIG. 23 provides a schematic of the rat ApoE locus and denotes with greybars the cutting site for ZFN1 and ZFN2. The cutting site for ZFN1 is int exon 3 and the cutting site for ZNF2 is in intron 3. The exactposition of the both ZFN sites is set forth in Table 16. The 5′ and 3′homology arms (45 kb and 23 kb, respectively) are denoted by the darkgrey boxes. Exon 1 of the ApoE gene is non-coding and is shown as anopen box closest to the 5′ homology arm. The three introns of the ApoEgene are denoted as lines. Exons 2 and 3 comprise coding regions and areshown as stippled grey boxes. Exon 4 contains both coding and non-codingsequences as denoted by the stippled grey shading and the open box.

The LTVEC employed was the same as that in Example 3.2(b)(ii) and shownin FIG. 22. The ZFNs were introduced as two expression plasmids, one foreach half of the ZFN pair. 20 ug of the plasmid for ZFN 1 and 20 ug ofthe plasmid for ZFN2 was used. ZFNs were purchased from Sigma. Theexpression of each ZFN was driven by the CMV promoter.

The targeting vector was electroporated into the rat ES cells obtainedin Example 1 and the cells were plated on 15 cm 2× dense neoR MEFs in2i+10 uM ROCKi. The transformed rat ES cells were cultured, selected,and maintained as described in Example 1.

As shown in Table 23, 288 colonies were screened and 16 targeted cloneswere obtained. The targeting efficiency was 5.56%. One clone wasinjected into blastocysts as described herein in Example 2.

Moreover, the employment of ZFN1 and ZFN2 produced one biallelictargeted clone, with an efficiency of 0.35%.

3.3(a): Targeting of the Rat Interleukin-2 Receptor Gamma (IL2r-γ) Locus

The rat Interleukin-2 receptor gamma (IL2r-γ) locus was targeted todisrupt IL2r-γ function. IL2r-γ plays an important role for signaling byIL-2, IL-4, IL-7, IL-9, IL-15, IL-21 and mutations in IL2r-γ areassociated with severe defects in T, B and NK cell development.

Targeting of the IL2r-γ locus was done using a targeting vectorcomprising an eGFP-hUb-neo cassette flanked with a 5′ and 3′ homologyarms homologous to the IL2r-γ locus. FIG. 26 depicts the genomicstructure of the rat IL2r-γ locus in which the IL2r-γ locus has beendisrupted by a 3.2 kb deletion. The targeted IL2r-γ locus also comprisedan eGFP gene and a self-deleting cassette containing Crei operablylinked to a mouse Protaminel promoter and a drug selection cassettecomprising a hUb promoter operably linked to a neomycin resistance gene.

Targeting efficiency at the IL2r-γ locus was determined and shown inTable 17. Linearized vector was electroporated into DA.2B rat ESCs, andtransfected colonies were cultured using standard techniques. Individualcolonies were picked and screened using a Loss of Allele (LOA) assay.

TABLE 17 rat IL2r-γ Targeting Efficiency Colonies Targeting Cell lineVector picked Targeted efficiency (%) DA.2B II2rg-floxed neo 136 1 0.7DA.2B II2rg-mSDC 96 4 4.2

Chimera production and germline transmission using IL2r-γ-targeted ratESC clones was performed. IL2r-γ-targeted rat ESC clones weremicroinjected into SD blastocysts, which were then transferred topseudopregnant SD recipient females, using standard techniques. Chimeraswere identified by coat color; male F0 chimeras were bred to SD females.Germline F1 pups were genotyped for the presence of the targeted IL2r-γallele (Table 18).

TABLE 18 Microinjection Results Exp Clone pups Chimeras 1 Il2rg-AA1 5 2(90, 70) 2 Il2rg-AA1 10 3 (90, 90, 80)

The phenotype of Il2rg^(−/Y) chimera #3 was further studied. Theperipheral blood mononuclear cells (PBMCs) were stained with antibodiesthat recognize antigens in several lymphoid lineages. GFP-positive PBMCswere detected from 2 of the chimeras. Moreover, the GFP+ cells werenegative for the T-cell marker CD3, and were mostly negative for theB-cell marker B220 and the NK cell marker CD161a. See, FIG. 30. Thesmall double-positive populations are consistent with the publishedIl2rg knockout phenotype in mice. These data were obtained from achimeric rat, which contains IL2 receptor gamma-positive cells, and thismay complicate the analysis of the phenotype.

3.3(b): Targeted Modification of the Rat Interleukin-2 Receptor Gamma(IL2r-γ) Locus

The rat Interleukin-2 receptor gamma (IL2r-γ) locus was targeted todisrupt the IL2r-γ function in rats. FIG. 26 shows the genomic structureof the rat Il2rg locus and the targeting vector introduced into thelocus. eGFP was chosen as a reporter so that the immunophenotype of thegenetically modified rats could be examined using FACS. Theself-deleting cassette (hUb-Neo; Prm1-Cre) was used to delete the drugsection cassette and the Cre gene specifically in male germ cells of theF0 rat. Additionally, the targeting vector was designed to delete theentire coding region (about 3.2 kb) of the rat Il2rg gene.

The size of the deletion in rat ESCs was confirmed by PCR using primersspecific to the rat Il2rg locus. Upon microinjection of the targetedclones into host embryos at a blastocyst stage, high percentage ofchimeras were obtained. Those chimeras have been set up for breeding. Todetermine if the targeting worked as expected, the peripheral blood fromthe chimeras were collected prior to breeding, and the phenotype of theimmune cells in the peripheral blood was analyzed via FACS. As shown inFIG. 30, GFP-positive cells were detected in the peripheral blood in 2of the 3 chimeras examined (upper right panel), and the chimeric ratscontained less than 1% of T cells, less than 1% of B cells, and lessthan 1% of NK-cells, which are positive for GFP (i.e., Il2rg KO cells).

3.4(a). Targeting the Rag2 Locus in Rats with a Large Targeting Vector(LTVEC)

Table 19 provides a summary of the genomic organization of the rat Rag2locus and the positions shown were taken from build 5.0 of the ReferenceSequence of the rat genome (ENSMBL). Rag2 is on chromosome 3 on the (+)strand.

TABLE 19 Genomic organization summary of the rat Rag2 locus. FeatureStart End length Notes Exon 1 97,851,317 97,851,448 132 Exon 297,854,635 97,854,693 59 Exon 3 97,858,260 97,859,615 1,356 containsentire coding sequence ATG 97,856,286 97,856,288 3 start codon TGA97,857,867 97,857,869 3 stop codon Rag2 97,856,289 97,859,784 3,496deletion

FIG. 27 provides a schematic of the rat Rag2 locus and a large targetingvector (LTVEC). The upper schematic of FIG. 27 shows the genomicorganization of the rat ApoE locus and the genomic regions correspondingto the 5′ and 3′ homology arms (48 Kb and 15 Kb, respectively; dark greyboxes). Rag2 comprises a single exon denoted by the stippled greyshading.

The lower schematic in FIG. 27 is the LTVEC. The 5′ and 3′ homology arms(48 kb and 15 kb, respectively) are denoted by the dark grey boxes. TheLTVEC comprises a reporter gene (lacZ) and a self-deleting cassetteflanked by loxP sites (open arrows). The self-deleting cassettecomprises a mouse Prm1 promoter operably linked to the Crei gene and adrug selection cassette comprising a human ubiquitin promoter operablylinked to a neomycin resistance gene. The Crei comprises two exonsencoding the Cre recombinase are separated by an intron (Crei) toprevent its expression in a prokaryotic cell. See, for example, U.S.Pat. No. 8,697,851 and U.S. Application Publication 2013-0312129, whichdescribe the self-deleting cassette in detail and are herebyincorporated by reference in their entirety. By employing a mouse Prm1promoter, the self-deleting cassette can be deleted specifically in malegerm cells of F0 rats.

The LTVEC was electroporated into the rat ES cells obtained in Example 1and the cells were plated on 15 cm 2× dense neoR MEFs in 2i+10 uM ROCKi.The transformed rat ES cells were cultured and maintained as describedin Example 1. Colonies are screened as described elsewhere herein andtargeted clones are obtained. The targeted clones are then injected intoa host embryo as described elsewhere herein to produce an F0 rat.

3.4. (b). Targeting the Rag1 and the Rag 2 Locus in Rats

FIG. 28 provides the genomic structure of the rat Rag1/Rag2 locus. CDSdenotes the coding sequence and grey boxes represent exons. Rag2 is onthe “plus” strand with transcription to the right. Rag1 is on the“minus” strand with transcription to the left. Mbp=million base pairs.

Table 20 provides a summary of the genomic organization of the rat Rag2and Rag1 locus and the positions shown were taken from build 5.0 of theReference Sequence of the rat genome (ENSMBL). Rag1 is on chromosome 3on the (−) strand.

TABLE 20 Genomic organization summary of the rat Rag1 locus. FeatureStart End length Notes Exon 1 97,877,145 97,877,066 80 Exon 2 97,872,50397,866,047 6,457 contains entire coding sequence ATG 97,872,48997,872,487 3 start codon TAA 97,869,369 97,869,367 3 stop codon Rag1-297,856,289 97,872,486 16,198 deletion

FIG. 29 provides a schematic of the rat Rag2 and Rag1 locus and a largetargeting vector (LTVEC). The upper schematic of FIG. 29 shows thegenomic organization of the Rag1 and Rag2 loci and the genomic regionscorresponding to the 5′ and 3′ homology arms (48 kb and 84 kb,respectively; dark grey boxes). Rag2 and Rag 1 each comprises a singleexon denoted by the stippled grey shading. The lower schematic in FIG.29 is the LTVEC. The 5′ and 3′ homology arms (48 kb and 84 kb,respectively) are denoted by the dark grey boxes. The LTVEC comprises areporter gene (lacZ) and a self-deleting cassette flanked by loxP sites(open arrows). The self-deleting cassette comprises a rat Prm1 promoteroperably linked to the Crei gene and a drug selection cassettecomprising a human ubiquitin promoter operably linked to a neomycinresistance gene. The Crei comprises two exons encoding the Crerecombinase are separated by an intron (Crei) to prevent its expressionin a prokaryotic cell. See, for example, U.S. Pat. No. 8,697,851 andU.S. Application Publication 2013-0312129, which describes theself-deleting cassette in detail and is hereby incorporated by referencein their entirety. By employing a rat Prm1 promoter that drivesexpression of Crei specifically in male germ cells, the self-deletingcassette can be deleted from the male germ cells of F0 rats.

The LTVEC was electroporated into the rat ES cells obtained in Example 1and the cells were plated on 15 cm 2× dense neoR MEFs in 2i+10 uM ROCKi.The transformed rat ES cells were cultured and maintained as describedin Example 1.

Colonies are screened as described elsewhere herein and targeted clonesare obtained. The targeted clones are then injected into a host embryoas described elsewhere herein to produce an F0 rat.

Example 4. Humanization

4.1. Humanization of Rat Genomic Loci

Humanization of rat genomic loci is carried out employing the rat EScells described herein, which are capable of sustaining theirpluripotency following one or more electroporations in vitro, and arecapable of transmitting the targeted genetic modifications to subsequentgenerations. In addition, in order to circumvent the limitations ofplasmids in accommodating a large genomic DNA fragment, and to overcomethe low efficiency of introducing a targeted genetic modification intoan endogenous locus in rat ES cells, one or more targeted geneticmodifications are carried out in bacteria, e.g., E. coli, by utilizingbacterial homologous recombination (BHR) and employing a large targetingvector (LTVEC). The LTVEC described herein, for example, includes alarge fragment of an endogenous rat genomic sequence with one or moremodifications or comprises an exogenous nucleic acid (e.g., a homologousor orthologous human nucleic acid) flanked with rat homology armscomplementary to specific genomic regions.

4.2. Humanization of Rat Immunoglobulin Loci

Humanization of an endogenous rat immunoglobulin heavy chain locus iscarried out by removing one or more endogenous rat immunoglobulin heavychain nucleic acid sequences (e.g., one or more endogenous V_(H) genesegments, one or more human D gene segments, and one or more human J_(H)gene segments); and introducing into the modified immunoglobulin locus atargeting vector, e.g., a large targeting vector (LTVEC) comprising: (i)one or more unrearranged human variable region nucleic acid sequences(e.g., one or more human V_(H) gene segments, one or more human D genesegments, and one or more human J_(H) gene segments), or one or morerearranged human variable region nucleic acid sequences (e.g., one ormore human rearranged V-D-J gene segments); (ii) a selection cassette(e.g., neomycin resistance gene flanked with loxP sites); and (iii) 5′and 3′ rat homology arms.

Briefly, one or more endogenous rat immunoglobulin heavy chain variableregion gene segments (i.e., one or more V_(H) gene segments, one or morehuman D gene segments, and one or more human J_(H) gene segments) in arat BAC clone are removed or inactivated by targeting the endogenous ratimmunoglobulin heavy chain locus with a selection cassette flanked byrat homology arms. More specifically, a targeting vector is constructedto contain a selection cassette (e.g., a neomycin resistance geneflanked with loxP sites) flanked with 5′ and 3′ rat homology arms thatare complementary to target rat genomic sequences (e.g., upstream anddownstream rat genomic DNA sequences encompassing one or more rat V_(H)gene segments, one or more human D gene segments, and one or more humanJ_(H) gene segments).

Next, bacterial cells containing a large rat genomic DNA fragmentencompassing a rat immunoglobulin heavy chain locus are selected andintroduced with a plasmid (e.g., pABG) encoding a recombinase operablylinked to a transiently inducible promoter. The targeting vectorconstructed above is then introduced into the recombination-competentbacterial cells. Following electroporation, the bacterial cells aretreated with an inducer (e.g., arabinoside) to initiate homologousrecombination between the targeting vector and the target rat genomicsequence in the BAC clone. Transformed cells are plated at a highdensity and subjected to drug selection to find colonies that aredrug-resistant. Drug-resistant colonies are picked and screened for thetargeted modification.

In order to facilitate identification of the targeted geneticmodification, a high-throughput quantitative assay, namely, modificationof allele (MOA) assay, is employed, which allows a large-scale screeningof a modified allele(s) in a parental chromosome following a geneticmodification. The MOA assay can be carried out via various analyticaltechniques, including, but not limited to, a quantitative PCR, e.g., areal-time PCR (qPCR). For example, the real-time PCR comprises a firstprimer set that recognizes the target locus and a second primer set thatrecognizes a non-targeted reference locus. In addition, the primer setcan comprise a fluorescent probe that recognizes the amplified sequence.Alternatively, the quantitative assay can be carried out via a varietyof analytical techniques, including, but not limited to,fluorescence-mediated in situ hybridization (FISH), comparative genomichybridization, isothermic DNA amplification, quantitative hybridizationto an immobilized probe(s), Invader Probes®, MMP Assays®, TaqMan®Molecular Beacon, and Eclipse™ probe technology. (See, for example,US2005/0144655, incorporated by reference herein in its entirety).

The bacterial cells comprising the modified rat BAC clone, i.e., a BACclone containing a rat genomic DNA sequence wherein one or moreendogenous heavy chain variable region gene segments (V_(H), D, and/orJ_(H) gene segments) have been deleted or inactivated, are thenelectroporated with a large targeting vector (LTVEC) comprising: (i) oneor more unrearranged human variable region nucleic acid sequences (e.g.,one or more unrearranged human V_(H) gene segments, one or more human Dgene segments, and one or more human J_(H) gene segments), or one ormore rearranged human variable region nucleic acid sequences (e.g., oneor more rearranged human V-D-J gene segments).

Initiation of homologous recombination in the bacterial cells and theselection of positive clones are performed as described above. Theunrearranged or rearranged human immunoglobulin heavy chain variableregion nucleic acid sequences, when targeted into the endogenousimmunoglobulin heavy chain locus, become operably linked to anendogenous rat immunoglobulin heavy chain constant region nucleic acidsequence. Alternatively, endogenous rat heavy chain constant regionlocus can be inactivated, for example, by deleting one or more rat heavychain constant region gene segments (CH) from the endogenous heavy chainconstant region locus, and can be replaced with a human heavy chainconstant region nucleic acid sequence.

Likewise, humanization of an endogenous rat immunoglobulin κ or λ lightchain locus is carried out by removing one or more endogenous ratimmunoglobulin and/or λ light chain variable region nucleic acidsequences (e.g., one or more endogenous κ rat V_(κ) gene segments andone or more endogenous rat J_(κ) gene segments); and targeting themodified immunoglobulin light chain locus with a targeting vector, e.g.,a large targeting vector (LTVEC), comprising: (i) one or moreunrearranged human immunoglobulin light chain variable region nucleicacid sequences (e.g., one or more human V_(κ) gene segments and one ormore human J_(κ) gene segments), or one or more rearranged humanvariable region nucleic acid sequences (e.g., one or more humanrearranged V_(κ)-J_(κ) gene segments); (ii) a selection cassette (e.g.,neomycin resistance gene flanked with loxP sites); and (iii) 5′ and 3′rat homology arms.

The unrearranged or rearranged human immunoglobulin light chain variableregion nucleic acid sequences, when targeted into the endogenousimmunoglobulin light chain locus, become operably linked to theendogenous rat immunoglobulin light chain constant region nucleic acidsequence.

The LTVEC so produced in the bacterial cells comprises, for example, aninsert nucleic acid that contains a humanized rat immunoglobulin heavychain or light chain locus in which one or more endogenous rat heavy orlight chain variable region gene segments have been replaced with one ormore human heavy or light chain variable region gene segments; and rathomologous arms (e.g., ranging from 5 kb to 150 kb) complementary tospecific genomic target sequences. The LTVEC comprising the geneticmodification described above is then linearized and electroporated intothe rat ES cells. Electroporated rat ES cells are plated at a highdensity to select drug-resistant ES cells comprising the targetingvector. The drug selection process removes the majority of the platedcells (˜99%), leaving behind individual colonies, each of which is aclone derived from a single cell. Of the remaining cells, most cells(˜80-100%) contain the targeting vector integrated at a random locationin the genome. Therefore, the colonies are picked and genotypedindividually in order to identify rat ES cells comprising the targetingvector at the correct genomic location (e.g., using the modification ofallele (MOA) assay described above).

In order to increase the efficiency of the targeted geneticmodification, the rat ES cells are electroporated with expressionvectors (or mRNA) that express ZFNs 1 and 2 (or TALENs 1 and 2) togetherwith the LTVEC. The targeting vector's homology arms lie outside the ZFNtarget site, therefore, the targeting vector is not cleaved by the ZFNs.The double strand break produced by the ZFNs stimulateshomology-directed repair (HDR), which otherwise accounts for a verysmall percentage of repairs occurred normally in mammalian cells(compared to non-homologous end-joining; NHEJ).

Alternatively, expression vectors containing a type II CRISPR-associatednuclease (e.g., Cas9), a guide RNA (including CRISPR-RNA (cr-RNA) andtrans-activating CRISPR RNA (tracrRNA)), as described herein, can beintroduced into the bacterial cells together with the LTVEC to increasethe efficiency of homologous recombination at the target genomic locus.Electroporated cells are plated at a high density and subjected to drugselection to find colonies that are drug-resistant. Drug-resistantcolonies are picked and screened for the targeted modification using themodification of allele (MOA) assay as described herein. Following theseprocedures, improvement in the targeting efficiency can be achieved. Forexample, the amount of improvement can be small (e.g., improve from 10%to 15%) or large (e.g., improve from 10% to 80%).

The selected rat ES cells comprising the targeted genetic modificationare then introduced into a host rat embryo, for example, a pre-morulastage or blastocyst stage rat embryo, and implanted in the uterus of asurrogate mother to generate a founder rat (F0 rat). Subsequently, thefounder rat is bred to a wild-type rat to create F1 progeny heterozygousfor the genetic modification. Mating of the heterozygous F1 rat canproduce progeny homozygous for the genetic modification.

4.3(a). Replacing Rat IL2rg with Human IL2 Receptor Gamma

Table 21 provides a summary of the genomic organization of the ratInterleukin 2 receptor gamma locus and the positions shown were takenfrom build 5.0 of the Reference Sequence of the rat genome (ENSMBL).IL2rg is on chromosome X on the (−) strand.

TABLE 21 Summary of the genomic organization of the rat Il2rg locusFeature Start End length Notes Exon 1 72,021,388 72,021,516   129contains ATG ATG 72,017,500 72,017,502     3 start codon Exon272,021,007 72,021,160   154 ZFN1a binding site 72,021,014 72,021,028   15 CAGGCCCTGAACCGC (SEQ ID NO: 17) ZFN1 cutting site 72,021,00872,021,013     6 TTCTGG (SEQ ID NO: 18) ZFN1b binding site 72,020,99372,021,007    15 GATTACCTGCGCTGGG (SEQ ID NO: 20) Exon3 72,020,60672,020,790   185 Exon4 72,020,274 72,020,413   140 Exon5 72,019,66272,019,824   163 Exon6 72,019,101 72,019,197    97 Exon7 72,018,84472,018,910    67 Exon8 72,017,856 72,018,506   651 contains TGA TGA72,018,321 72,018,323     3 stop codon Il2rg deletion 72,018,32372,021,502 3,180

The lower schematic in FIG. 26 is the targeting vector for the IL2rg 3.2kb deletion. The targeting vector comprises a reporter gene (eGFP)operably linked to the endogenous promoter and a self-deleting cassetteflanked by loxP sites (open arrows). The self-deleting cassettecomprises the Crei gene operably linked to a mouse Prm1 promoter and aselection cassette comprising a neomycin resistance gene operably linkedto a human ubiquitin promoter.

The Crei gene comprises two exons encoding a Cre recombinase, which areseparated by an intron (Crei) to prevent its expression in a prokaryoticcell. See, See, for example, U.S. Pat. No. 8,697,851 and U.S.Application Publication 2013-0312129, which describe the self-deletingcassette in detail and are hereby incorporated by reference in theirentirety. By employing the mouse Prm1 promoter the Cre expressioncassette and the drug selection cassette can be deleted specifically inmale germ cells of F0 rats. The targeting vector was electroporated intothe rat ES cells obtained in Example 1 and the cells were plated on 15cm 2× dense neomycin-resistant MEFs in 2i+10 uM ROCKi. The transformedrat ES cells were cultured, selected, and maintained as described inExample 1.

As shown in Table 23, 168 colonies were screened and 6 targeted cloneswere obtained. The targeting efficiency was 3.57%.

Clones are injected into blastocysts as described herein in Example 1.Clones producing F0 rats are obtained and F0 rats that transmit thetargeted modification through the germline are obtained.

Example 4.3(b). Replacing Rat IL2rg Ecto-Domain with Human IL2rgEcto-Domain

The full-length humanization of IL 2 receptor gamma is useful becauserats having this modified locus will produce human Il2rg; and this wouldallow for the detection of human Il2rg in rats with antibodies specificto human Il2rg.

The ecto-humanization (i.e., replacing the rat ecto-domain of Il2rg withthe human ecto-domain of Il2rg) will result in an Il2rg polypeptide thatwill bind the human ligands for Il2rg, but because the cytoplasmicdomain is still rat, it ecto-humanized form of Il2rg will also interactwith the rat signaling machinery. FIG. 33 provides a sequence alignmentof the human IL-2rg protein (SEQ ID NO: 20; NP_000197.1); the rat IL-2rgprotein (SEQ ID NO: 21; NP_543165.1); and the chimeric IL-2rg protein(SEQ ID NO: 22) comprising the human ecto-domain of IL-2rg fused to theremainder of the rat IL-2rg protein. The junction between the human andrat IL-2rg is noted by the vertical line.

Table 22 provides a summary of the genomic organization of the ratInterleukin 2 receptor gamma locus and the positions shown were takenfrom build 5.0 of the Reference Sequence of the rat genome (ENSMBL).IL2rg is on chromosome X on the (−) strand. Further noted is theposition of the ecto-domain of IL2rg.

TABLE 22 Summary of the genomic organization of the rat Il2rg locusFeature Start End length Notes Exon 1 71,111,444 71,111,543 100 containsATG ATG 71,111,537 71,111,539 3 start codon Exon2 71,110,897 71,111,050154 Exon3 71,110,504 71,110,688 185 Exon4 71,110,156 71,110,295 140Exon5 71,109,228 71,109,390 163 Exon6 71,108,599 71,108,645 47 containstransmembrane domain Exon7 71,108,277 71,108,346 70 Exon8 71,107,40471,107,921 518 contains TGA TGA 71,108,736 71,108,738 3 stop codonfull-length 71,107,404 71,111,539 4,136 (ATG to TGA humaniza- plus 3′poly-A) tion: ecto- 71,108,679 71,111,539 2,861 (ATG to beginninghumaniza- of transmembrane tion domain)

A plasmid targeting vectors were constructed to replace the ratecto-domain of the interleukin 2 receptor gamma coding region with thehuman ecto domain as shown in FIG. 31. The targeting vector waselectroporated into the rat ES cells obtained in Example 1 and the cellswere plated on 15 cm 2× dense neomycin-resistant MEFs in 2i+10 uM ROCKi.The transformed rat ES cells were cultured, selected, and maintained asdescribed in Example 1.

As shown in Table 23, 192 colonies were screened and 13 targeted cloneswere obtained. The targeting efficiency was 6.77%.

Clones are injected into blastocysts as described herein in Example 1.Clones producing F0 rats are obtained and F0 rats that transmit thetargeted modification through the germline are obtained.

Example 5. Summary

TABLE 23 Summary of rat targeting with various vector types and nucleaseagents discussed in Examples 3 and 4. Table 23 Targeting Summary ClonesClones transmitting Colonies Targeted Targeting Biallelic BiallelicClones producing through Example # Locus Vector screened Clonesefficiency targeted efficiency Injected chimeras germline Notes3.2(a)(ii) ApoE plasmid 384 23 5.99% 3 3 2 3.2(a)(iii) ApoE + plasmid384 290 75.52% 8 2.08% 2 2 1 These 2 clones are ZFN biallelic targeted3.3(a) Il2rg plasmid 232 5 2.16% 6 5 3.2(b)(ii) ApoE LTVEC 288 8 2.78% 10.35% 3 1 LTVEC 3.2(b)(iii) ApoE LTVEC 288 16 5.56% 1 0.35% 1 N/A Thisclone is biallelic LTVEC + targeted ZFN 4.3(a) Il2rg plasmid 168 6 3.57%replaces entire rat Human- Il2rg with human Il2rg ization 1 4.3(b) Il2rgplasmid 192 13 6.77% 2 N/A replaces rat Il2rg ecto- Human- domain withhuman ization 2 Il2rg ecto-domain 3.4(a) Rag2 LTVEC 270 N/A Predicted5.7 KB deletion 3.4(b) Rag1-2 LTVEC 256 N/A Predicted 16.2 kb deletion

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. Unless otherwise apparentfrom the context of any embodiment, aspect, step or feature of theinvention can be used in combination with any other. Reference to arange includes any integers within the range, any subrange within therange. Reference to multiple ranges includes composites of such ranges.

1.-36. (canceled)
 37. A method for making a genetically modified rat pluripotent stem cell clone capable of generating a genetically modified rat comprising a targeted genetic modification and transmitting the targeted genetic modification through the germline, comprising: (a) culturing a population of pluripotent rat cells on a layer of feeder cells that is not modified to express leukemia inhibitory factor (LIF) with a medium comprising about 50 U/mL to about 150 U/mL LIF, and a combination of inhibitors consisting of a MEK inhibitor and a GSK3 inhibitor; (b) introducing into the pluripotent rat cells a large targeting vector (LTVEC) comprising an insert nucleic acid flanked by a 5′ homology arm homologous to a first nucleic acid sequence at a genomic locus of interest and a 3′ homology arm homologous to a second nucleic acid sequence at the genomic locus of interest to produce the targeted genetic modification via homologous recombination, wherein the sum total of the 5′ and the 3′ homology arms is at least 10 kb; and (c) obtaining a pluripotent rat cell clone comprising the targeted genetic modification at the genomic locus of interest, wherein the obtaining consists of identifying in a single cloning step a pluripotent rat cell clone comprising the targeted genetic modification at the genomic locus of interest and capable of generating a genetically modified rat comprising the targeted genetic modification and transmitting the targeted genetic modification through the germline.
 38. The method of claim 37, wherein the pluripotent rat cells are rat embryonic stem (ES) cells.
 39. The method of claim 38, wherein the rat ES cells or male (XY) rat ES cells or female (XX) rat ES cells.
 40. The method of claim 38, wherein the rat ES cells are diploid and/or form spherical, free-floating colonies in culture.
 41. The method of claim 37, wherein the pluripotent rat cells are derived from a DA strain or an ACI strain.
 42. The method of claim 37, wherein the pluripotent rat cells are characterized by one or more of the following characteristics: (I) expression of at least one pluripotency marker selected from the group consisting of Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, and Utf1; (II) lack of expression of one or more of the pluripotency markers c-Myc, Ecat1, and Rexo1; (III) lack of expression of one or more of the mesodermal markers Brachyury and Bmpr2; (IV) lack of expression of one or more of the endodermal markers Gata6, Sox17, and Sox7; (V) lack of expression of one or more of the neural markers Nestin and Pax6.
 43. The method of claim 37, wherein the concentration of LIF in the medium is between about 75 U/mL to about 125 U/mL.
 44. The method of claim 43, wherein the concentration of LIF in the medium is between about 90 U/mL to about 110 U/mL.
 45. The method of claim 44, wherein the concentration of LIF in the medium is about 100 U/mL.
 46. The method of claim 37, wherein the LIF is a mouse LIF.
 47. The method of claim 37, wherein the concentration of the MEK inhibitor is 0.8 μM to about 1.2 μM, and the concentration of the GSK3 inhibitor is about 2.5 μM to about 3.5 μM.
 48. The method of claim 47, wherein the concentration of the MEK inhibitor is about 1 μM, and the concentration of the GSK3 inhibitor is about 3 μM.
 49. The method of claim 45, wherein the concentration of the MEK inhibitor is about 1 μM and the concentration of the GSK3 inhibitor is 3 μM.
 50. The method of claim 37, wherein the MEK inhibitor is PD0325901 and the GSK3 inhibitor is CHIR99021.
 51. The method of claim 37, wherein the feeder cells comprise mitotically inactivated mouse embryonic fibroblasts.
 52. The method of claim 37, wherein the feeder cells comprise mitotically inactivated mouse embryonic fibroblasts, and the medium is a 2i medium comprising DMEM/F12 basal medium at a concentration of 1×; neurobasal medium at a concentration of 1×; penicillin/streptomycin at a concentration of 1%; L-Glutamine at a concentration of 4 mM; 2-mercaptoethanol at a concentration of 0.1 mM; N2 supplement at a concentration of 1×; B27 supplement at a concentration 1×; LIF at a concentration of 100 U/mL; PD0325901 at a concentration of 1 μM, and CHIR99021 at a concentration of 3 μM.
 53. The method of claim 37, wherein: (I) the sum total of the 5′ and the 3′ homology arms of the LTVEC is from 10 kb to about 150 kb, (II) the 5′ homology arm ranges from about 5 kb to about 100 kb or the 3′ homology arm ranges from about 5 kb to about 100 kb; or (III) the LTVEC is from about 20 kb to about 400 kb.
 54. The method of claim 37, wherein the insert nucleic acid is from about 5 kb to about 400 kb.
 55. The method of claim 37, wherein the insert nucleic acid: (i) comprises a polynucleotide of interest comprising a genomic nucleic acid sequence that encodes a human immunoglobulin heavy chain variable region amino acid sequence; (ii) comprises a polynucleotide of interest comprising a genomic nucleic acid sequence that encodes a human immunoglobulin light chain variable region amino acid sequence; (iii) comprises a polynucleotide of interest comprising a polynucleotide encoding at least a region of a T cell receptor; or (iv) comprises a polynucleotide of interest comprising at least one disease allele.
 56. The method of claim 55, wherein the insert nucleic acid comprises a polynucleotide of interest comprising a polynucleotide encoding at least a region of a T cell receptor alpha.
 57. The method of claim 37, wherein the insert nucleic acid: (i) is from a human; (ii) comprises a knock-in allele of at least one exon of an endogenous gene; (iii) comprises a regulatory element; (iv) comprises a conditional allele; (v) comprises a nucleic acid flanked by site-specific recombination target sequences; (vi) comprises a polynucleotide encoding a selection marker; or (vii) comprises a reporter gene operably linked to a promoter.
 58. The method of claim 57, wherein the insert nucleic acid comprises a polynucleotide encoding a selection marker and/or a reporter gene flanked by site-specific recombination target sequences or comprises a self-deleting selection cassette.
 59. The method of claim 37, wherein introducing step (b) further comprises introducing a nucleic acid encoding a nuclease agent that promotes homologous recombination between the LTVEC and the genomic locus of interest in the pluripotent rat cells.
 60. The method of claim 59, wherein the nuclease agent comprises: (I) a chimeric protein comprising a zinc finger-based DNA binding domain fused to a FokI endonuclease; (II) a chimeric protein comprising a Transcription Activator-Like Effector Nuclease (TALEN); or (III) a CRISPR/Cas system.
 61. The method of claim 60, wherein the nuclease agent comprises the CRISPR/Cas system, wherein the CRISPR/Cas system comprises a Cas9 nuclease and a guide RNA.
 62. The method of claim 61, wherein the guide RNA comprises the sequence set forth in SEQ ID NO: 2, 3, 4, 5, 6, 7, or
 8. 63. The method of claim 37, wherein the targeted genetic modification is biallelic.
 64. The method of claim 37, wherein the targeted genetic modification comprises deletion of an endogenous rat nucleic acid sequence, wherein the deletion ranges from about 5 kb to about 3 Mb.
 65. The method of claim 37, wherein the targeted genetic modification comprises insertion of an exogenous nucleic acid sequence ranging from about 5 kb to about 400 kb.
 66. The method of claim 37, wherein the targeted genetic modification comprises: (I) replacement of an endogenous rat nucleic acid sequence with an exogenous sequence; (II) replacement of an endogenous rat nucleic acid sequence with a homologous or an orthologous nucleic acid sequence; (III) deletion of an endogenous rat nucleic acid sequence; (IV) insertion of an exogenous nucleic acid sequence comprising a nucleic acid sequence that is homologous or orthologous to an endogenous rat nucleic acid sequence; (V) insertion of a chimeric nucleic acid sequence comprising a human nucleic acid sequence and a rat nucleic acid sequence; (VI) insertion of a conditional allele flanked by site-specific recombinase target sequences; or (VII) insertion of a reporter gene operably linked to a promoter active in a rat cell.
 67. The method of claim 37, wherein the targeted genetic modification comprises: (I) an insertion of the insert nucleic acid, wherein the insert nucleic acid comprises a human nucleic acid sequence; (II) a replacement of a rat nucleic acid sequence at the genomic locus of interest with a human nucleic acid sequence, wherein the replaced rat nucleic acid sequence is homologous or orthologous to the human nucleic acid sequence; (III) a chimeric nucleic acid sequence comprising a human nucleic acid sequence and a rat nucleic acid sequence; or (IV) a combination thereof.
 68. The method of claim 67, wherein the size of the insertion is from about 5 kb to about 500 kb, or wherein the size of the replacement is from about 5 kb to about 500 kb.
 69. The method of claim 37, wherein the genomic locus of interest comprises: (i) an Il2rg locus, an ApoE locus, a Rag1 locus, a Rag2 locus, or a Rag2/Rag1 locus; (ii) an immunoglobulin locus; or (iii) a T cell receptor locus.
 70. The method of claim 69, wherein the immunoglobulin locus is an immunoglobulin heavy chain locus or an immunoglobulin light chain locus.
 71. The method of claim 70, wherein the immunoglobulin light chain locus is a λ or κ immunoglobulin light chain locus.
 72. The method of claim 69, wherein the genomic locus of interest comprises a T cell receptor alpha locus.
 73. The method of claim 37, wherein the introducing step is mediated by electroporation.
 74. The method of claim 37, wherein steps (a)-(c) are sequentially repeated to allow for the targeted integration of at least two insert nucleic acids into the genomic locus of interest.
 75. The method of claim 37, wherein a modification-of-allele assay is used to identify the pluripotent rat cell clone comprising the targeted genetic modification at the genomic locus of interest.
 76. The method of claim 37, further comprising: (d) introducing the pluripotent rat cell clone into a rat host embryo; (e) gestating the rat host embryo comprising the pluripotent rat cell clone in a surrogate mother, wherein the surrogate mother produces an F0 progeny genetically modified rat comprising the targeted genetic modification; and breeding the F0 progeny genetically modified rat with another rat to produce an F1 progeny genetically modified rat comprising the targeted genetic modification, wherein the targeted genetic modification is transmitted through the germline. 